WO2010076766A1 - Genes associated with plant tiller number and uses thereof - Google Patents

Genes associated with plant tiller number and uses thereof Download PDF

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Publication number
WO2010076766A1
WO2010076766A1 PCT/IB2009/055988 IB2009055988W WO2010076766A1 WO 2010076766 A1 WO2010076766 A1 WO 2010076766A1 IB 2009055988 W IB2009055988 W IB 2009055988W WO 2010076766 A1 WO2010076766 A1 WO 2010076766A1
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dlt
plant
nucleic acid
sequence
protein
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PCT/IB2009/055988
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French (fr)
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Chu Chengcai
Tong Hongning
Yun Jin
Wenbo Liu
Feng Li
Jun Fang
Lihuang Zhu
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Institute Of Genetics And Developmental Biology
Syngenta Participations Ag
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Publication of WO2010076766A1 publication Critical patent/WO2010076766A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8298—Brassinosteroids

Definitions

  • the invention relates generally to compositions and methods for altering plant tiller number using dwarf and low tillering (DLT) genes.
  • the aforementioned compositions include polynucleotides, polypeptides, vectors and host cells.
  • the present invention also relates to plants transformed by the aforementioned compositions and methods.
  • Brassinosteroids are an important class of phytohormones involved in various processes during plant growth and development. Brassinosteroids play a significant role in controlling the height and bending angle of the lamina joint of a plant. A plant's height directly correlates with its anti-lodging ability, and the bending angle of the lamina joint is associated with planting density and the efficiency with which plants capture sunlight. A compact morphology can greatly reduce the overshadowing between leaf blades, thus enhancing the capacity of each leaf to capture sunlight. This improves the collective photosynthetic capability of the population and leads to increased crop yield.
  • brassinosteroids may also improve tillering and the transport of assimilation products of photosynthesis from source to sink, subsequently improving the grain filling of rice seeds ⁇ see Wu, et al., Plant Cell, 20:2130-2145).
  • the OsD WARF4-dcF ⁇ cicnt mutant osdwarf4- 1 has a distinctly smaller lamina joint bending angle and is particularly suitable for close planting.
  • osdwarf4-l mutants can improve yield by 32% under dense planting conditions (44.4 plants/m 2 ) without any extra fertilizer ⁇ see Sakamoto et al., Nat. BiotechnoL, 24:105-109 (2006)).
  • OsBRIl loss-of-function mutants show a range of phenotypes, and the d61-l and d61-2 alleles produce agronomically useful traits such as semidwarf stature, erect leaves, and elongated neck internodes.
  • Two transgenic OsBRIl knock-down lines (BKDl 1 and BKD22) had grain yields that were calculated to be respectively 35% and 26% larger than the corresponding wild type plants planted at high density ⁇ see Morinaka et al., Plant Physiol., 141:924-931 (2006)).
  • the present invention relates to isolated dwarf and low-tillering (DLT) polynucleotides, polypeptides, vectors and host cells expressing isolated DLT polynucleotides capable of conferring desirable properties to plants, including altering tiller number.
  • DLT dwarf and low-tillering
  • the isolated DLT polynucleotides provided herein include nucleic acids comprising (a) a nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence of SEQ ID NO: 3; (c) a nucleotide sequence at least 70% identical to (a) or (b); (c) those that specifically hybridize to the complement of (a) or (b) under stringent hybridization conditions; (d) an open reading frame encoding a protein comprising a polypeptide sequence of SEQ ID NO: 2 or 4; (e) an open reading frame encoding a protein comprising a polypeptide sequence at least 70% identical to
  • SEQ ID NO: 2 or 4 SEQ ID NO: 2 or 4; and (f) a nucleotide sequence that is the complement of any one of (a)-(f).
  • the isolated DLT polypeptides provided herein include (a) the amino acid sequence of SEQ ID NO: 2 or 4, (b) an amino acid sequence derived from SEQ ID NO: 2 or 4 by substitution and/or deletion and/or addition of one or more amino acid residues wherein the amino acid sequence is capable of altering tiller number and (c) an amino acid sequence at least
  • the host cells provided herein include those comprising the isolated polynucleotides and vectors of the present invention.
  • the host cell can be from an animal, plant, or microorganism, such as E. coli. Plant cells are particularly contemplated.
  • the host cell can be isolated, excised, or cultivated.
  • the host cell may also be part of a plant.
  • the present invention further relates to a plant or a part of a plant that comprises a host cell of the present invention. Rice is particularly contemplated.
  • the present invention also relates to the transgenic seeds of the plants.
  • the present invention further relates to a method for producing a plant comprising regenerating a transgenic plant from a host cell of the present invention, or hybridizing a transgenic plant of the present invention to another non-transgenic plant. Plants produced by these methods are also encompassed by the present invention, and rice is particularly contemplated.
  • the present invention further relates to methods of altering a trait in a plant or part of a plant using the isolated polynucleotides, polypeptides, constructs and vectors of the present invention.
  • These traits include altering (i.e., increasing or decreasing) tiller number in comparison to a corresponding wild type plant and dwarf stature.
  • these traits are altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant.
  • the present invention further relates to the use of the isolated polynucleotides, polypeptides, constructs and vectors of the present invention to alter tiller number in a plant.
  • tiller number is altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant.
  • DLT nucleic acids or proteins of the invention such as SEQ ID NOs: 1-4
  • Figure 1 shows the phenotype of dlt mutant and wild type (WT) rice at (a) the vegetative phase and (b) the productive phase.
  • Figure 2(a) is a linkage map of DLT.
  • DLT is located between S240 and S1551 on chromosome 6, near the 5' telomere. Sequence-tagged site markers are named according to their chromosome physical positions, and relevant recombinant numbers are indicated above the linkage map.
  • PAC Pl -derived artificial chromosome
  • BAC BAC clones in the dlt mutation candidate region are shown under the linkage map.
  • Figure 2(b) shows the structure of the putative DL T full-length cDNA. The position of the mutation is shown. The underlined and italicized TGA is the putative new stop codon of an aberrant DLT in the dlt mutant.
  • Figure 2(c) is an RT-PCR of DL T. The band sizes of DLT are indicated. ACTINl was used as a control.
  • Figure 2(d) is a protein sequence alignment of DLT (OsGRAS32) and AtGRAS8 with AtGAI, AtRGAl, and AtSCR using Cluster W. The arrow indicates the mutation site. Five conserved motifs specific to GRAS proteins are indicated (leucine heptad I, VHIID, leucine heptad I, PFYRE, and SAW motifs). conserveed amino acids are highlighted in black and gray.
  • Figure 3 demonstrates the results of phenotypic complementation by introduction of the DLT gene into dlt mutants.
  • Figures 3 (a) and 3(b) show the gross morphology at (a) the vegetative phase and (b) the productive phase of WT, transgenic (dlt-c) and mutant plants.
  • Figure 3(c) is a PCR of WT, transgenic (dlt-c) and mutant plants. Arrows indicate band size.
  • Figure 4(a) shows the lamina joint bending response to various amounts of 24-epiBL by the micro-drop method.
  • Figure 4(b) shows the lamina joint bending response to 5 ng/ml 24- epiBL by the excised leaf segment method.
  • Figure 4(c) shows the coleoptile elongation response to 0.1 ⁇ M 24-epiBL.
  • Figure 5(a) shows the expression of DLT in various organs analyzed by quantitative RT-PCR analysis. Panicles and culms were collected when they had reached 1 cm length. The SAM, root, and the third leaf sheath and blade were harvested from 2-week-old plants.
  • Figure 5(b) shows the expression pattern of DLT in culm tissues. Intl to Int5 represent five internodes counted from top to bottom. The lowest zones of each internode were collected to extract RNA.
  • Figure 6 shows the GUS staining of PRO DLT :GUS transgenic line tissues. Pane 1 shows a longitudinal section after 3 days germination. The arrow indicates the SAM.
  • Pane 2 shows a longitudinal section of 7-day-old seedling after germination in dark. A primary root is also shown. The arrow indicates the SAM.
  • Pane 3 shows an unexpanded fourth leaf without greening from a 2-week old seedling.
  • Pane 4 shows the microscopic observation of a cross- section of pane 3.
  • Pane 5 shows a third leaf from a 2-week-old seedling. The arrow indicates the lamina joint.
  • Pane 6 shows a longitudinal section of a young culm. The arrow indicates the shoot apex.
  • Pane 7 shows a full-length elongating uppermost internode.
  • Pane 8 shows a full- length elongated third internode.
  • Pane 9 is a cross-section of the lowest part of pane 7.
  • Pane 10 is amagnified image of part of pane 9.
  • Pane 11 shows a young spikelet.
  • Pane 12 shows an older floret.
  • Pane 13 shows a young root with lateral root protruding.
  • Pane 14 shows a lateral root.
  • Scale bars 1 mm (panes 1-3, 5, 6, 9, and 11-14), 100 ⁇ m (panes 4 and 10) or 1 cm ( panes 7 and 8).
  • Figure 7(a) is a quantitative RT-PCR analysis of transcription levels for DLT at various times after exogenous 1 ⁇ M 24-epiBL treatment.
  • Figure 7(b) shows increased expression of DLT in d.2-1 and dll-2 mutants assayed by quantitative RT-PCR analysis. Shiokari plants are used as the WT control, and WT expression is set at 1.0.
  • Figure 8 shows a comparison of brassinosteroid (BR)-related gene expression between WT and dlt mutants with or without 24-epiBL. Gene expression was normalized to that of the rice ACTINl gene, and levels in WT or levels without 24-epiBL treatment are set as 1.0.
  • BR brassinosteroid
  • Figure 8(a) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT 'in WT and the dlt mutant.
  • Figure 8(b) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT in WT and the dlt mutant grown on half-strength MS with or without 1 ⁇ M 24-epiBL.
  • Figure 8(c) shows the quantitative RT-PCR analysis of expression of BR downstream genes and signaling genes.
  • Figure 9 shows an electrophoretic mobility shift assay.
  • a WT DNA probe derived from the DLT promoter was incubated with 200 ng recombinant OsBZRl . Competition reactions with either unlabeled WT probe or the mutant (Mt) form were performed to demonstrate the specific binding of OsBZRl to the BRRE in the DLT promoter.
  • Figure 10(a) shows the relative level of DLT expression in ten transgenic To plants.
  • Figure 10(b) shows the differing phenotypes of wild type and transgenic T 0 plants.
  • Figure 11 is a statistical analysis of the tiller number often T 2 transgenic plant lines performed during the heading stage.
  • Plant line number is shown on the x-axis, while tiller number is shown on the y-axis. The mean tiller number and standard deviation are provided. Statistically significant differences generated from a Student's t-test are indicated at the P ⁇ 0.05 (*) and P ⁇ 0.01 (**) levels.
  • nucleic acid As used herein, the terms “nucleic acid”, “polynucleotide”, “polynucleotide molecule”, “polynucleotide sequence” and plural variants are used interchangeably to refer to a wide variety of molecules, including single strand and double strand DNA and RNA molecules, cDNA sequences, genomic DNA sequences of exons and introns, chemically synthesized DNA and RNA sequences, and sense strands and corresponding antisense strands. Polynucleotides of the invention may also comprise known analogs of natural nucleotides that have similar properties as the reference natural nucleic acid.
  • polypeptide As used herein, the terms “polypeptide”, “protein” and plural variants are used interchangeably and refer to a compound made up of a single chain of amino acids joined by peptide bonds.
  • Polypeptides of the invention may comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Polypeptides may include both L-form and D-form amino acids.
  • non-genetically encoded amino acids include but are not limited to 2- aminoadipic acid; 3-aminoadipic acid; ⁇ -aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2- aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N- ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N- methylvaline; norvaline; norleucine;
  • Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
  • Exemplary DLT polynucleotides of the invention are set forth as SEQ ID NOs: 1 and 2 and substantially identical sequences encoding proteins capable of altering the tiller number of a plant.
  • Exemplary DLT polypeptides of the invention are set forth as SEQ ID NOs: 2 and 4 and substantially identical proteins capable of altering the tiller number of a plant.
  • Substantially identical sequences are those that have at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • substantially identical nucleic acids or proteins perform substantially the same function.
  • substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population. An allelic difference may be as small as one base pair.
  • Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • a mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
  • Substantially identical nucleic acids are also identified as nucleic acids that hybridize specifically to or hybridize substantially to a reference sequence (e.g., SEQ ID NO: 1).
  • a reference sequence e.g., SEQ ID NO: 1
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol, 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI), or by visual inspection (see Ausubel et al., infra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences are substantially identical.
  • Stringent conditions are those under which a nucleic acid probe will typically hybridize to its target sequence but to no other sequences when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA).
  • Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments are both sequence- and environment-dependent. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York (1993).
  • highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42 0 C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72 0 C for about 15 minutes.
  • stringent wash conditions is a 0.2X SSC wash at 65 0 C for 15 minutes ⁇ see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is IX SSC at 45 0 C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4X - 6X SSC at 40 0 C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M sodium ions, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 0 C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • a substantially identical nucleotide sequence preferably hybridizes to a reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 2X SSC, 0.1% SDS at 50 0 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in IX SSC, 0.1% SDS at 50 0 C, still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 0.5X SSC, 0.1% SDS at 50 0 C, even more preferably in 7% sodium dode
  • nucleic acid sequences or proteins are substantially identical are that the that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents, or are immunologically cross-reactive with, or specifically bind to, each other. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This may occur, for example, when two nucleotide sequences comprise conservatively substituted variants as permitted by the genetic code.
  • This also includes degenerate codon substitutions wherein the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acids Res., 19:5081(1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); and Rossolini et al. MoI. Cell Probes, 8:91-98 (1994)).
  • both the polynucleotides and the polypeptides of the present invention may be conservatively substituted at one or more residues.
  • conservative amino acid substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • Nucleic acids of the invention also comprise nucleic acids complementary to SEQ ID NOs: 1 and 3, and subsequences and elongated sequences of SEQ ID NOs: 1 and 3 and complementary sequences thereof.
  • Complementary sequences are two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs.
  • complementary sequences maybe substantially similar to one another as described previously.
  • a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
  • a subsequence is a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence.
  • An exemplary subsequence is a probe or a primer.
  • An elongated sequence is one in which nucleotides (or other analogous molecules) are added to a nucleic acid sequence.
  • a polymerase e.g., a DNA polymerase
  • the nucleotide sequence may be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, introns, additional restriction enzyme sites, multiple cloning sites, and other coding segments.
  • the present invention also provides vectors comprising the disclosed nucleic acids, including vectors for recombinant expression, wherein a nucleic acid of the invention is operatively linked to a functional promoter.
  • a promoter is in functional combination with the nucleic acid such that the transcription of the nucleic acid is controlled and regulated by the promoter region.
  • Vectors refer to nucleic acids capable of replication in a host cell, such as plasmids, cosmids, and viral vectors.
  • Polynucleotides of the present invention may be cloned, synthesized, altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art. Site-specific mutagenesis to create base pair changes, deletions, or small insertions is also known in the art (see e.g., Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual.
  • Isolated polypeptides of the invention may be purified and characterized using a variety of standard techniques that are known to the skilled artisan (see e.g., Schroder et al., The Peptides, 1965, Academic Press, New York; Bodanszky, Principles of Peptide Synthesis, 2nd rev. ed. 1993, Springer- Verlag, Berlin/ New York; Ausubel (ed.), Short Protocols in Molecular Biology. 3rd ed., 1995, Wiley, New York).
  • the present invention also encompasses methods for detecting a nucleic acid molecule that encodes a DLT protein. Such methods may be used to detect gene variants or altered gene expression. Sequences detected by methods of the invention may detected, subcloned, sequenced, and further evaluated by any measure well known in the art using any method usually applied to the detection of a specific DNA sequence. Thus, the nucleic acids of the present invention may be used to clone genes and genomic DNA comprising the disclosed sequences. Alternatively, the nucleic acids of the present invention may be used to clone genes and genomic DNA of related sequences.
  • Levels of a DL T nucleic acid molecule may be measured, for example, using an RT-PCR assay (see e.g., Chiang, J Chromatogr. A., 806:209- 218 (1998) and references cited therein).
  • the present invention also encompasses genetic assays using DLT nucleic acids for quantitative trait loci (QTL) analysis and to screen for genetic variants, for example by allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sd. USA, 80(l):278-282 (1983)), oligonucleotide ligation assays (OLAs) (Nickerson et al., Proc. Natl. Acad. ScL USA, 87(22):8923-8927 (1990)), single-strand conformation polymorphism (SSCP) analysis (Orita et al., Proc. Natl. Acad. Sci.
  • ASO allele-specific oligonucleotide
  • OVAs oligonucleotide ligation assays
  • SSCP single-strand conformation polymorphism
  • the present invention also encompasses functional fragments of a DLT polypeptide, for example, fragments that have the ability to alter tiller number similar to that of SEQ ID NOs: 2 and 4. Functional polypeptide sequences that are longer than the disclosed sequences are also encompassed.
  • one or more amino acids may be added to the N-terminus or C- terminus of an antibody polypeptide.
  • additional amino acids may be employed in a variety of applications, including but not limited to purification applications. Methods of preparing elongated proteins are known in the art.
  • the present invention also encompasses methods for detecting a polypeptide. Such methods can be used, for example, to determine levels of protein expression and correlate the level of expression with the presence or change in phenotype, trait, or level of expression in a different gene or gene product.
  • the method involves an immunochemical reaction with an antibody that specifically recognizes a protein.
  • An expression system refers to a host cell comprising a heterologous nucleic acid and the protein encoded by the heterologous nucleic acid.
  • a heterologous expression system may comprise a host cell transfected with a construct comprising a DLT nucleic acid encoding a protein operatively linked to a promoter, or a cell line produced by introduction of DLT nucleic acids into a host cell genome.
  • the expression system may further comprise one or more additional heterologous nucleic acids relevant to DLT function, such as targets of DLT transcriptional activation or repression activity. These additional nucleic acids may be expressed as a single construct or multiple constructs.
  • a construct for expressing a DLT protein may include a vector sequence and a DLT nucleotide sequence, wherein the DL T nucleotide sequence is operatively linked to a promoter sequence.
  • a construct for recombinant DL T expression may also comprise transcription termination signals and sequences required for proper translation of the nucleotide sequence. Preparation of an expression construct, including addition of translation and termination signal sequences, is known to one skilled in the art.
  • the promoter may be any polynucleotide sequence which shows transcriptional activity in the chosen plant cells, plant parts, or plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Where the promoter is native or endogenous to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence of the invention, the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
  • the promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic.
  • promoters Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley et al., Nucleic Acids Res., 15:2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized ⁇ see e.g., Roberts et al., Proc. Natl. Acad. Sd. USA, 76:760-4 (1979)). Many suitable promoters for use in plants are well known in the art. [0055] For example, suitable constitutive promoters for use in plants include the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PClSV) promoter (U.S. Patent No.
  • PClSV peanut chlorotic streak caulimovirus
  • Suitable inducible promoters for use in plants include the promoter from the ACEl system which responds to copper (Mett et al., Proc. Natl. Acad. ScL USA, 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., MoI. Gen. Genetics, 227:229-237 (1991); and Gatz et al., MoI. Gen. Genetics, 243:32-38 (1994)); and the promoter of the Tet repressor from TnIO (Gatz et al., MoI. Gen. Genet.
  • Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond.
  • An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. ScL USA, 88:10421 (1991)) or the recent application of a chimeric transcription activator, XVE, for use in an estrogen receptor-based inducible plant expression system activated by estradiol (Zuo et al., Plant J., 24:265-273 (2000)).
  • inducible promoters for use in plants are described in EP 332104, PCT International Publication Nos. WO 93/21334 and WO 97/06269. Promoters composed of portions of other promoters and partially or totally synthetic promoters can also be used ⁇ see e.g., Ni et al., Plant J., 7:661-676 (1995) and PCT International Publication No. WO 95/14098 describing such promoters for use in plants).
  • Tissue-specific or tissue-preferential promoters useful for the expression of the novel DLT genes of the invention in plants including the cotton rubisco promoter disclosed in U.S. Patent No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent No. 5,604,121; and the cestrum yellow leaf curling virus promoter disclosed in PCT International Publication No. WO 01/73087.
  • Chemically inducible promoters useful for directing the expression of DLT genes in plants are disclosed in U.S. Patent No. 5,614,395.
  • the promoter may include, or be modified to include, one or more enhancer elements to thereby provide for higher levels of transcription.
  • Suitable enhancer elements for use in plants include the PClSV enhancer element (U.S. Patent No. 5,850,019), the CaMV 35S enhancer element (U.S. Patent Nos. 5,106,739 and 5,164,316) and the FMV enhancer element (Maiti et al., Transgenic Res., 6:143-156 (1997)). See also PCT International Publication No. WO 96/23898.
  • Such constructs can contain a 'signal sequence' or 'leader sequence' to facilitate co- translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted.
  • the construct can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
  • a signal sequence is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation.
  • a leader sequence refers to any sequence that, when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Plant expression cassettes may also contain an intron, such that mRNA processing of the intron is required for expression.
  • Such constructs can also contain 5 ' and 3 ' untranslated regions.
  • a 3 ' untranslated region is a polynucleotide located downstream of a coding sequence.
  • Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions.
  • a 5' untranslated region is a polynucleotide located upstream of a coding sequence.
  • the termination region may be native with the transcriptional initiation region, may be native with the sequence of the present invention, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A.
  • tumefaciens such as the octopine synthase and nopaline synthase termination regions (see e.g., Guerineau et al., MoI. Gen. Genet., 262:141-144 (1991); Proudfoot, Cell, 64:671-674 (1991); Sanfacon et al., Genes Dev., 5:141-149 (1991); Mogen et al., Plant Cell, 2:1261-1272 (1990); Munroe et al., Gene, 91:151-158 (1990); Ballas et al., Nucleic Acids Res., 17:7891-7903 (1989); and Joshi et al., Nucleic Acid Res., 15:9627-9639 (1987)).
  • the vector and DLT sequences may be optimized for increased expression in the transformed host cell. That is, the sequences can be synthesized using host cell-preferred codons for improving expression, or may be synthesized using codons at a host- preferred codon usage frequency. Generally, the GC content of the polynucleotide will be increased ⁇ see e.g., Campbell et al., Plant Physiol, 92:1-11 (1990) for a discussion of host- preferred codon usage). Methods are known in the art for synthesizing host-preferred polynucleotides ⁇ see e.g., U.S. Patent Nos.
  • polynucleotides of interest are targeted to the chloroplast for expression.
  • the expression cassette may additionally contain a polynucleotide encoding a transit peptide to direct the nucleotide of interest to the chloroplasts.
  • transit peptides are known in the art (see e.g., Von Heijne et al., Plant MoI. Biol. Rep., 9:104-126 (1991); Clark et al., J. Biol.
  • the polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons (see e.g., U.S. Patent No.
  • a plant expression cassette i.e., a DLT open reading frame operatively linked to a promoter
  • a plant transformation vector which allows for the transformation of DNA into a cell.
  • Such a molecule may consist of one or more expression cassettes, and may be organized into more than one vector DNA molecule.
  • binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens et al., Trends in Plant Science, 5:446-451 (2000)).
  • a plant transformation vector comprises one or more DNA vectors for achieving plant transformation.
  • DNA vectors for achieving plant transformation.
  • These vectors are often referred to in the art as binary vectors.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a polynucleotide of interest (i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired).
  • a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border)
  • a selectable marker that is engineered to be capable of expression in a plant cell
  • a polynucleotide of interest i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired.
  • different antibiotic or herbicide selectable markers may be preferred.
  • Selection markers used routinely in transformation include the nptllgene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, Gene, 19:259-268 (1982); and Bevan et al., Nature, 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res., 18:1062 (1990), and Spencer et al., Theor. Appl. Genet., 79:625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, MoI. Cell.
  • sequences required for bacterial replication are sequences required for bacterial replication.
  • the cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein.
  • the selectable marker sequence and the sequence of interest are located between the left and right borders.
  • a second plasmid vector contains the transacting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as in understood in the art (Hellens et al., 2000).
  • a nucleotide sequence of the present invention is directly transformed into a plastid genome.
  • a major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter.
  • Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT International Application Publication WO 95/16783, and in McBride et al., Proc. Natl. Acad. Sci. USA, 91:7301-7305 (1994).
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526-8530 (1990); Staub et al., Plant Cell, 4:39-45 (1992)). This results in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.
  • a nucleotide sequence of the present invention is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • Host cells are cells into which a heterologous nucleic acid molecule of the invention may be introduced.
  • Representative eukaryotic host cells include yeast and plant cells, as well as prokaryotic hosts such as E.coli and B. subtilis.
  • Preferred host cells for functional assays substantially or completely lack endogenous expression of a DLT protein.
  • a host cell strain may be chosen which modulates the expression of the recombinant sequence, or modifies and processes the gene product in a specific manner. For example, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins).
  • Appropriate cell lines or host cells may be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system may be used to produce a non-glycosylated core protein product, and expression in yeast will produce a glycosylated product.
  • the present invention further encompasses recombinant expression of a DLT protein in a stable cell line.
  • Methods for generating a stable cell line following transformation of a heterologous construct into a host cell are known in the art ⁇ see e.g., Joyner, Gene Targeting: A Practical Approach. 1993, Oxford University Press, Oxford/New York).
  • transformed cells, tissues, and plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny or propagated forms thereof.
  • the present invention also provides DLT knockout plants comprising a disruption of a
  • DLT ⁇ ocMS DLT ⁇ ocMS.
  • a disrupted gene may result in expression of an altered level of full-length DLT protein or expression of a mutated variant DLT protein (e.g., SEQ ID NO: 4).
  • Plants with complete or partial functional inactivation of the DLT gene may be generated, e.g., by expressing a mutant DL T allele in the plant.
  • a knockout plant in accordance with the present invention may also be prepared using anti-sense, double-stranded RNA, or ribozyme DL T constructs, driven by a universal or tissue-specific promoter to reduce levels of DLT gene expression in somatic cells, thus achieving a "knock-down" phenotype.
  • the present invention also provides the generation of plants with conditional or inducible inactivation of DLT.
  • the present invention also encompasses transgenic plants with specific "knocked-in” modifications in the disclosed DLT gene, for example to create an over-expression mutant having a dominant negative phenotype.
  • "knocked-in” modifications include the expression of mutant alleles of the DLT gene.
  • DL T knockout plants may be prepared in monocot or dicot plants, such as maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.
  • monocot or dicot plants such as maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach,
  • a plant refers to a whole plant, a plant organ (e.g., root, stem, leaf, flower bud, or embryo), a seed, a plant cell, a propagule, an embryo, other plant parts (e.g., protoplasts, pollen, pollen tubes, ovules, embryo sacs, zygotes) and progeny of the same.
  • Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
  • introduction of a polynucleotide into plant cells is accomplished by one of several techniques known in the art, including but not limited to electroporation or chemical transformation ⁇ see e.g., Ausubel, ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Indiana). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test polynucleotide sequence) from non-transformed cells (those not containing or not expressing the test polynucleotide sequence). In one aspect of the invention, genes are useful as a marker to assess introduction of DNA into plant cells.
  • Stable transformation refers to introduction of a polynucleotide construct into a plant such that it integrates into the genome of the plant and is capable of being inherited by progeny thereof.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (i.e., temperature and/or herbicide). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (see e.g., Hiei et al., Plant J., 6:271-282 (1994); and Ishida et al., Nat. Biotechnol, 14:745-750 (1996)). A general description of the techniques and methods for generating transgenic plants are found in Ayres et al., CRC Crit. Rev.
  • Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods to transfer DNA (see e.g., Hiei et al., Plant J., 6:271-282 (1994); Ishida et al., Nat. Biotechnol., 14:745-750 (1996); Ayres et al., CRC Crit. Rev. Plant ScL, 13:219-239 (1994); and Bommineni et al., Maydica, 1997, 42:107-120 (1997)).
  • the first method is co-cultivation of Agrobacterium with cultured isolated protoplasts. This method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • the second method is transformation of cells or tissues with Agrobacterium. This method requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
  • the third method is transformation of seeds, apices or meristems with Agrobacterium. This method requires micropropagation. [0082] The efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art.
  • transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles ⁇ see e.g., Bidney et al., Plant Molec. Biol, 18:301-313 (1992).
  • the plant cells are transfected with vectors via particle bombardment (i.e., with a gene gun).
  • particle bombardment i.e., with a gene gun.
  • Particle mediated gene transfer methods are known in the art, are commercially available, and include, but are not limited to, the gas driven gene delivery instrument described in U.S. Patent No. 5,584,807. This method involves coating the polynucleotide sequence of interest onto heavy metal particles, and accelerating the coated particles under the pressure of compressed gas for delivery to the target tissue.
  • Other particle bombardment methods are also available for the introduction of heterologous polynucleotide sequences into plant cells.
  • these methods involve depositing the polynucleotide sequence of interest upon the surface of small, dense particles of a material such as gold, platinum, or tungsten.
  • the coated particles are themselves then coated onto either a rigid surface, such as a metal plate, or onto a carrier sheet made of a fragile material such as mylar.
  • the coated sheet is then accelerated toward the target biological tissue.
  • the use of the flat sheet generates a uniform spread of accelerated particles that maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the polynucleotide sample into the target tissue.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide of interest, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers that are appropriate for the particular cell system that is used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ., 20:125 (1994)).
  • the cells that have been transformed may be grown into plants in accordance with conventional ways ⁇ see e.g., McCormick et al., Plant Cell Rep., 5:81-84 (1986)). These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as transgenic seed) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • Transgenic plants of the invention can be homozygous for the added polynucleotides; i.e., a transgenic plant that contains two added sequences, one sequence at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains the added sequences according to the invention, germinating some of the seed produced and analyzing the resulting plants produced for enhanced enzyme activity (i.e., herbicide resistance) and/or increased plant yield relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous polynucleotides. Selfing of appropriate progeny can produce plants that are homozygous for all added, exogenous polynucleotides that encode a polypeptide of the present invention. Back-crossing to a parental plant and outcrossing with a non-transgenic plant are also contemplated. [0089] Following introduction of DNA into plant cells, the transformation or integration of the polynucleotide into the plant genome is confirmed by various methods such as analysis of polynucleotides, polypeptides and metabolites associated with the integrated sequence. [0090] DLT Inhibitors
  • the present invention further discloses assays to identify DLT binding partners and DLT inhibitors.
  • DLT antagonists/inhibitors are agents that alter chemical and biological activities or properties of a DLT protein. Methods of identifying inhibitors involve assaying a reduced level or quality of DLT function in the presence of one or more agents. Exemplary DLT inhibitors include small molecules as well as biological inhibitors as described herein below.
  • the term "agent” refers to any substance that potentially interacts with a DLT nucleic acid or protein, including any of synthetic, recombinant, or natural origin. An agent suspected to interact with a protein may be evaluated for such an interaction using the methods disclosed herein.
  • Exemplary agents include but are not limited to peptides, proteins, nucleic acids, small molecules (e.g., chemical compounds), antibodies or fragments thereof, nucleic acid- protein fusions, any other affinity agent, and combinations thereof.
  • An agent to be tested may be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.
  • a small molecule refers to a compound, for example an organic compound, with a molecular weight of less than about 1,000 daltons, more preferably less than about 750 daltons, still more preferably less than about 600 daltons, and still more preferably less than about 500 daltons.
  • a small molecule also preferably has a computed log octanol-water partition coefficient in the range of about -A to about +14, more preferably in the range of about -2 to about +7.5.
  • exemplary nucleic acids that may be used to disrupt DLT function include antisense RNA and small interfering RNAs (siRNAs) ⁇ see e.g., U.S. Application Publication No. 20060095987).
  • inhibitory molecules may be prepared based upon the DLT gene sequence and known features of inhibitory nucleic acids ⁇ see e.g., Van der Krol et al., Plant Cell, 2:291- 299 (1990); Napoli et al., Plant Cell, 2:279-289 (1990); English et al., Plant Cell, 8:179-188 (1996); and Waterhouse et al., Nature Rev. Genet, 2003, 4:29-38 (2003).
  • Agents may be obtained or prepared as a library or collection of molecules.
  • a library may contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more.
  • a molecule may comprise a naturally occurring molecule, a recombinant molecule, or a synthetic molecule.
  • a plurality of agents in a library may be assayed simultaneously.
  • agents derived from different libraries may be pooled for simultaneous evaluation.
  • Representative libraries include but are not limited to a peptide library (U.S. Patent Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Patent Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Patent Nos.
  • a library may comprise a random collection of molecules.
  • a library may comprise a collection of molecules having a bias for a particular sequence, structure, or conformation, for example, as for inhibitory nucleic acids (see e.g., U.S. Patent Nos. 5,264,563 and 5,824,483).
  • Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. patents cited herein above. Numerous libraries are also commercially available.
  • a control level or quality of DLT activity refers to a level or quality of wild type DLT activity, for example, when using a recombinant expression system comprising expression of SEQ ID NOs: 1 or 3.
  • a control level or quality of DLT activity comprises a level or quality of activity in the absence of the agent.
  • a control level may also be established by a phenotype or other measureable trait.
  • Assaying the inhibiting capacity of an agent may comprise determining a level of DLT gene expression; determining DNA binding activity of a recombinantly expressed DLT protein; determining an active conformation of a DLT protein; or determining a change in a trait in response to binding of a DLT inhibitor (e.g., increased or decreased tiller number).
  • a method of identifying a DLT inhibitor may comprise (a) providing a cell, plant, or plant part expressing a DLT protein; (b) contacting the cell, plant, or plant part with an agent; (c) examining the cell, plant, or plant part for a change in a trait as compared to a control; and (d) selecting an agent that induces a change in the trait as compared to a control. Any of the agents so identified in the disclosed inhibitory or binding assays (see hereinafter) may be subsequently applied to a cell, plant or plant part as desired to effectuate a change in that cell, plant or plant part.
  • the present invention also encompasses a rapid and high throughput screening method that relies on the methods described herein.
  • This screening method comprises separately contacting a DLT protein with a plurality of agents.
  • the plurality of agents may comprise more than about 10 4 samples, or more than about 10 5 samples, or more than about 10 6 samples.
  • the in vitro and cellular assays of the invention may comprise soluble assays, or may further comprise a solid phase substrate for immobilizing one or more components of the assay.
  • a DLT protein, or a cell expressing a DLT protein may be bound directly to a solid state component via a covalent or non-covalent linkage.
  • the binding may include a linker molecule or tag that mediates indirect binding of a DLT protein to a substrate.
  • the present invention also encompasses methods of identifying of a DLT inhibitor by determining specific binding of a substance (e.g., an agent described previously) to a DLT protein.
  • a method of identifying a DLT binding partner may comprise: (a) providing a DLT protein of SEQ ID NO: 2 or 4; (b) contacting the DLT protein with one or more agents under conditions sufficient for binding; (c) assaying binding of the agent to the isolated DLT protein; and (d) selecting an agent that demonstrates specific binding to the DLT protein.
  • Specific binding may also encompass a quality or state of mutual action such that binding of an agent to a DLT protein is inhibitory.
  • Specific binding refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials.
  • the binding of an agent to a DLT protein may be considered specific if the binding affinity is about IxIO 4 M "1 to about 1x10 6 M "1 or greater.
  • Specific binding also refers to saturable binding.
  • Scatchard analysis may be carried out as described, for example, by Mak et al, J Biol. Chem., 264:21613-21618 (1989).
  • Several techniques may be used to detect interactions between a DLT protein and an agent without employing a known competitive inhibitor.
  • Representative methods include, but are not limited to, Fluorescence Correlation Spectroscopy, Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Spectroscopy, and BIACORE® technology, each technique described herein below. These methods are amenable to automated, high-throughput screening.
  • FCS Fluorescence Correlation Spectroscopy
  • the sample size may be as low as 10 3 fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium.
  • the diffusion rate is a function of the mass of the molecule and decreases as the mass increases.
  • FCS may therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding.
  • the target to be analyzed e.g., a DLT protein
  • a sequence tag such as a poly-histidine sequence, inserted at the N-terminus or C-terminus.
  • the expression is mediated in a host cell, such as E.coli, yeast, Xenopus oocytes, or mammalian cells.
  • the protein is purified using chromatographic methods.
  • the poly-histidine tag may be used to bind the expressed protein to a metal chelate column such as Ni 2+ chelated on iminodiacetic acid agarose.
  • the protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPYTM reagent (available from Molecular Probes of Eugene, Oregon).
  • the protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood of New York, New York). Ligand binding is determined by changes in the diffusion rate of the protein.
  • SELDI Surface-Enhanced Laser Desorption/Ionization
  • a target protein e.g., a DLT protein
  • the target protein is bound to a SELDI chip either by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • a chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler).
  • the chip is then washed in solutions of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF.
  • Ligands that specifically bind a target protein are identified by the stringency of the wash needed to elute them.
  • BIACORE® relies on changes in the refractive index at the surface layer upon binding of a ligand to a target protein (e.g., a DLT protein) immobilized on the layer.
  • a target protein e.g., a DLT protein
  • a collection of small ligands is injected sequentially in a 2-5 microliter cell, wherein the target protein is immobilized within the cell. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface.
  • SPR surface plasmon resonance
  • the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein.
  • a target protein is recombinantly expressed, purified, and bound to a BIACORE® chip. Binding may be facilitated by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • a chip thus prepared is then exposed to one or more potential ligands via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler).
  • the SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics of on rate and off rate allows the discrimination between non-specific and specific interaction (see also Homola et al., Sensors and Actuators, 54:3-15 (1999) and references therein).
  • the present invention also encompasses methods of identifying DLT binding partners and inhibitors that rely on a conformational change of a DLT protein when bound by or otherwise interacting with a substance (e.g., an agent described previously). For example, application of circular dichroism to solutions of macromolecules reveals the conformational states of these macromolecules. The technique may distinguish random coil, alpha helix, and beta chain conformational states.
  • circular dichroism analysis may be performed using a recombinantly expressed DLT protein.
  • a DLT protein is purified, for example by ion exchange and size exclusion chromatography, and mixed with an agent. The mixture is subjected to circular dichroism.
  • the conformation of a DLT protein in the presence of an agent is compared to a conformation of a DLT protein in the absence of the agent.
  • a change in conformational state of a DLT protein in the presence of an agent identifies a DLT binding partner or inhibitor. Representative methods are described in U.S. Patent Nos. 5,776,859 and 5,780,242.
  • Antagonistic activity of the inhibitor may be assessed using functional assays, such assaying nitrate content, nitrate uptake, lateral root growth, or plant biomass, as described herein.
  • cells expressing DLT may be provided in the form of a kit useful for performing an assay of DLT function.
  • a kit for detecting a DLT may include cells transfected with DNA encoding a full-length DLT protein and a medium for growing the cells.
  • Assays of DLT activity that employ transiently transfected cells may include a marker that distinguishes transfected cells from non-transfected cells.
  • a marker may be encoded by or otherwise associated with a construct for DLT expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding DLT and the marker.
  • Representative detectable molecules that are useful as markers include but are not limited to a heterologous nucleic acid, a protein encoded by a transfected construct (e.g., an enzyme or a fluorescent protein), a binding protein, and an antigen.
  • Assays employing cells expressing recombinant DLT or plants expressing DLT may additionally employ control cells or plants that are substantially devoid of native DLT and, optionally, proteins substantially similar to a DLT protein.
  • a control cell When using transiently transfected cells, a control cell may comprise, for example, an untransfected host cell.
  • a control cell When using a stable cell line expressing a DLT protein, a control cell may comprise, for example, a parent cell line used to derive the -DZr-expressing cell line.
  • a method for producing an antibody that specifically binds a DLT protein.
  • a full-length recombinant DLT protein is formulated so that it may be used as an effective immunogen, and used to immunize an animal so as to generate an immune response in the animal.
  • the immune response is characterized by the production of antibodies that may be collected from the blood serum of the animal.
  • An antibody is an immunoglobulin protein, or antibody fragments that comprise an antigen binding site (e.g., Fab, modified Fab, Fab', F(ab') 2 or Fv fragments, or a protein having at least one immunoglobulin light chain variable region or at least one immunoglobulin heavy chain region).
  • Antibodies of the invention include diabodies, tetrameric antibodies, single chain antibodies, tretravalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and domain-specific antibodies that recognize a particular epitope. Cell lines that produce anti-DLT antibodies are also encompassed by the invention.
  • Specific binding of an antibody to a DLT protein refers to preferential binding to a DLT protein in a heterogeneous sample comprising multiple different antigens. Substantially lacking binding describes binding of an antibody to a control protein or sample, i.e., a level of binding characterized as non-specific or background binding.
  • the binding of an antibody to an antigen is specific if the binding affinity is at least about 10 ⁇ 7 M or higher, such as at least about 10 ⁇ 8 M or higher, including at least about 10 ⁇ 9 M or higher, at least about 10 "11 M or higher, or at least about 10 ⁇ 12 M or higher.
  • DLT antibodies prepared as disclosed herein may be used in methods known in the art relating to the expression and activity of DLT proteins, e.g., for cloning of nucleic acids encoding a DLT protein, immunopurification of a DLT protein, and detecting a DLT protein in a plant sample, and measuring levels of a DLT protein in plant samples.
  • an antibody of the present invention may further comprise a detectable label, including but not limited to a radioactive label, a fluorescent label, an epitope label, and a label that may be detected in vivo.
  • Methods for selection of a label suitable for a particular detection technique, and methods for conjugating to or otherwise associating a detectable label with an antibody are known to one skilled in the art.
  • dlt A dwarf and low tillering mutant (dlt) was identified from a library of rice T-DNA insertion mutants from the (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). As shown in Figure 1, the dlt mutant is 60% as tall and has only half the number of tillers of the corresponding Zhonghua 11 wild-type (WT) plants. In addition, the mutant plant has a compact morphology, erect leaves and panicles, reduced fertility, and a reduced ratio of the second internode to the total internode length. Microscopic observation also revealed that the dlt mutant had decreased cell length and less organized cellular morphology. These observations are consistent with plants that exhibit altered brassinosteroid signaling and/or synthesis.
  • DLT encodes a protein of 617 amino acids (DLT; SEQ ID NO: 2) that belongs to the plant-specific GRAS family.
  • the frameshift caused by the deletion generates an unrelated peptide (dlt; SEQ ID NO: 4) after amino acid 142 and a new stop codon (TGA) that is 2 bp downstream from the original stop codon (TAA) ⁇ see Figures 2(b) and (d)).
  • the mutation at amino acid 142 leads to loss of all conserved motifs, indicating that dlt is a knockout mutant ⁇ see Figure 2(d)).
  • RT-PCR analysis showed that aberrant DLT transcripts are expressed in the dlt plant ⁇ see Figure 2(c)).
  • a genomic fragment of 7577 bp containing the entire DLT coding sequence, 3253 bp of the 5' upstream region and 2471 bp of the 3' downstream region was digested from the BAC clone OSJNBa0038F22( Arizona Genomics Institute) using restriction enzymes BamHI and Kpnl (Promega). The fragment was recovered, ligated with a binary vector pCAMBIA1300 (Cambia, Australia), and transformed into Agrobacterium AGLl (ATCC). The transformed AGLl was used to infect the callus of a dit mutant as described, for example, in Yi et al., Journal of Genetics and Genomics, 28(4):352-358 (2001).
  • PCR amplification was performed on the genomic DNA of the regenerated plants using forward (5'- CATCAATCCATTGCAGGGACGAT-3' (SEQ ID NO: 5)) and reverse (5'- CGTTGAGCGTGAAGTGCAGGAA-S' (SEQ ID NO: 6) primers flanking the 62 bp deleted segment of the DLT gene. Forty positive transgenic plants were identified ⁇ see Figure 3(c)).
  • a third brassinosteroid response assay was performed to assess the effect of 24-epi- brassinolide on coleoptile elongation in dlt mutants (see e.g., Yamamuro et al., 2000). Seeds were germinated and grown on 0.7% agar medium supplemented with various concentrations of 24-epiBL. Comparison of coleoptile length showed that the dlt mutant has a much lower response to BL than WT does (see Figure 4(c)).
  • RNAs were extracted from various organs and tissues of the dlt mutant, using MMLV reverse transcriptase (Promega) to perform reverse transcription. Real-time fluorescence quantitative PCR was subsequently performed to detect the expression of DLT.
  • Rice ACTLNl was used as the internal control, and SYBR Green I was used as the dye.
  • Primers 5'- TGCGGATACTCAACGCCATC A-3' (forward; SEQ ID NO: 7) and 5'- ACTCGCCGACTCCGGTGATC-3' (reverse; SEQ ID NO: 8) were used to amplify DLT, and primers 5'-AGCAACTGGGATGATATGGA-S' (forward; SEQ ID NO: 9) and 5'-
  • CAGGGCGATGTAGGAAAGC-3' (reverse; SEQ ID NO: 10) were used to amplify ACTINl.
  • GUS staining showed that, in young seedlings, DLT is mainly expressed in the shoot apical meristem and elongating cells (Figure 6, panes 1 and 2). In leaves, DLT is expressed at significantly higher levels in unexpanded leaves than in green functional leaves, although with a slightly preferential expression in the leaf joint ( Figure 6, panes 3-5). DLT expression could not be detected in mature leaves by GUS staining.
  • DLT is expressed strongly in the internodes prior to rapid elongation
  • DLT also showed higher expression in the vascular cylinder and lateral root outgrowth locations, but much lower expression in cortex tissues ( Figure 6, panes 13 and 14). These expression patterns correlate well with DLTs putative function in cell elongation and division. Young tissues are those in which cells are actively dividing and elongating, and this is also where BR functions are most active.
  • d.2-1 is a dwarf mutant exhibiting a pleiotropic abnormal phenotype similar to that of the rice brassinosteroid-insensitive mutant, d61.
  • Hong et al. concluded that the D2 gene encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway ⁇ see Hong et al., Plant Cell, 15:2900-2910 (2003)).
  • dll-2 is a dwarf mutant that bears small round grains. Tanabe et al.
  • DIl gene also encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway ⁇ see Tanabe et al., Plant Cell, 17:776-790 (2005)).
  • the experiment was performed in triplicate. As shown in Figure 7(b), DLT expression in d.2-1 and dll-2 plants was significantly greater than in wild type Shiokari plants. The observations from these two brassinosteroid-synthesizing mutants further reinforce the conclusion that brassinosteroids negatively regulate the expression of DLT
  • Fluorescence quantitative PCR was used to determine the expression levels of several known brassinosteroid synthesizing genes ⁇ D2, DIl, OsCPD and OsBR ⁇ ox) and two genes
  • BRs brassinosteroids
  • BRREs brassinosteroid response elements
  • the OsBZRl coding region was cloned into a maltose binding protein (MBP) fusion vector (pET-MALc-H vector) using primers Os-BES lNAsp718 (5'-CTCGGTACCGGAGCTGGTGGGTATGACGTC-S'; SEQ ID NO: 13) and OsBESlCHind3 (5'-CGCAAGCTTTCATTTCGCGCCGACGCCGAGC-S'; SEQ ID NO: 14).
  • MBP-OsBZRl was purified from E. coli using amylose resin (NEB).
  • Wild type oligos derived from the DLT promoter, and mutant forms in which each nucleotide in a brassinosteroid response element (CGTGCG; SEQ ID NO: 15) was replaced with an adenosine were synthesized and annealed.
  • the WT probe was labeled with 32 P-C-ATP, and approximately 0.5 ng of probe was used for each binding assay. For competition experiments, excess unlabeled probe was added to the reactions at indicated molar ratios compared to labeled probe.
  • OsBZRl can bind to the labeled WT probe and unlabeled WT probe competes for binding with OsBRZl. OsBZRl does not bind to the mutant probe in which the BRRE is altered as described above. These results demonstrate that OsBZRl can bind to the DLT promoter through the BRRE.
  • the recombinant expression vector was transformed into the callus of the rice cultivar Zhonghua 11 via an Agrobacterium AGl (ATCC) mediated method. Ten transgenic T 0 generation plants were obtained by resistance screening.
  • Agrobacterium AGl Agrobacterium AGl
  • RNAs were extracted from the ten To generation transgenic positive plants and expression levels of DLT were determined by quantitative fluorescence PCR as described previously.
  • the DLT expression level in wild type rice Zhonghua 11 was set as 1 and the ratio of the DLT expression level in each of the transgenic plants to that of the wild type was calculated.
  • the experiments were performed in triplicate.
  • T 0 generation transgenic plants had significantly upregulated DLT gene expression. Of these seven plants, five of them had more than 100-fold increase in DL T expression levels as compared to their Zhonghua 11 counterparts. These five plants exhibited a curly leaf blade, enlarged lamina joint angle, increased tiller number and slight dwarfism (see plant no. 5 in Figure 10(b)). The other two T 0 transgenic plants, which had less than 100-fold increases in expression of DLT, exhibited significantly increased tiller numbers and slightly increased height, (see plant no. 10 in Figure 10(b)). These observations indicate that increased DLT expression leads to increased tiller number in rice.
  • Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110
  • Asn Ala lie Thr Pro lie Pro Arg Phe Leu His Phe Thr Leu Asn Glu 305 310 315 320
  • Trp Glu Ala Arg Phe Ala Arg Ala Leu Arg Tyr Tyr Ala Ala Ala Phe 465 470 475 480
  • Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110

Abstract

Compositions and methods for altering plant tiller number using dwarf and low tillering (DLT) genes.

Description

TITLE OF THE INVENTION [0001] Genes Associated with Plant Tiller Number and Uses Thereof
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority to Chinese Patent Application No. CN 200810247366. 3 filed 30 December 2008, herein incorporated by reference in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] The invention relates generally to compositions and methods for altering plant tiller number using dwarf and low tillering (DLT) genes. The aforementioned compositions include polynucleotides, polypeptides, vectors and host cells. The present invention also relates to plants transformed by the aforementioned compositions and methods.
BACKGROUND OF THE INVENTION
[0004] Brassinosteroids are an important class of phytohormones involved in various processes during plant growth and development. Brassinosteroids play a significant role in controlling the height and bending angle of the lamina joint of a plant. A plant's height directly correlates with its anti-lodging ability, and the bending angle of the lamina joint is associated with planting density and the efficiency with which plants capture sunlight. A compact morphology can greatly reduce the overshadowing between leaf blades, thus enhancing the capacity of each leaf to capture sunlight. This improves the collective photosynthetic capability of the population and leads to increased crop yield. Recently, researchers discovered that brassinosteroids may also improve tillering and the transport of assimilation products of photosynthesis from source to sink, subsequently improving the grain filling of rice seeds {see Wu, et al., Plant Cell, 20:2130-2145).
[0005] In last two decades, the synthesis and signaling pathways of brassinosteroids in Arabidopsis have been extensively researched, and multiple mechanisms modulate brassinosteroid signaling {see e.g., Gendron, J.M. and Wang, Z. Y., Curr. Opin. Plant Biol., 10:436-441 (2007) and Li, J. and Jin, H., Trends Plant ScL, 12:37-41 (2007)). To the contrary, these pathways are much less clear in rice, and only a handful of genes have been identified to this point: D2, DIl, OsDW ARF 4 and BRDl have been identified as members of the synthetic pathway, while OsBRI and OsBZRl appear to have predominant roles in the signaling pathway. [0006] However, it has been demonstrated that brassinosteroid-deficient mutants are associated with enhanced grain yield. For example, the OsD WARF4-dcFιcicnt mutant osdwarf4- 1 has a distinctly smaller lamina joint bending angle and is particularly suitable for close planting. In a field trial, it was discovered that osdwarf4-l mutants can improve yield by 32% under dense planting conditions (44.4 plants/m2) without any extra fertilizer {see Sakamoto et al., Nat. BiotechnoL, 24:105-109 (2006)).
[0007] Further, OsBRIl loss-of-function mutants (d61) show a range of phenotypes, and the d61-l and d61-2 alleles produce agronomically useful traits such as semidwarf stature, erect leaves, and elongated neck internodes. Two transgenic OsBRIl knock-down lines (BKDl 1 and BKD22) had grain yields that were calculated to be respectively 35% and 26% larger than the corresponding wild type plants planted at high density {see Morinaka et al., Plant Physiol., 141:924-931 (2006)).
[0008] Wu also reported that over-expression of sterol C-22 hydroxylases involved in brassinosteroid biosynthesis enhanced rice yield. Although the transgenic rice plants had greater lamina joint bending angles and were slightly taller than wild type plants, they also had increased tiller number, larger panicles and more seed per panicle {see Wu, et al., Plant Cell, 20:2130- 2145). In view of this type of evidence, the discovery of additional genes and gene products involved in brassinosteroid synthesis and signaling is highly desirable.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention relates to isolated dwarf and low-tillering (DLT) polynucleotides, polypeptides, vectors and host cells expressing isolated DLT polynucleotides capable of conferring desirable properties to plants, including altering tiller number. [0010] The isolated DLT polynucleotides provided herein include nucleic acids comprising (a) a nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence of SEQ ID NO: 3; (c) a nucleotide sequence at least 70% identical to (a) or (b); (c) those that specifically hybridize to the complement of (a) or (b) under stringent hybridization conditions; (d) an open reading frame encoding a protein comprising a polypeptide sequence of SEQ ID NO: 2 or 4; (e) an open reading frame encoding a protein comprising a polypeptide sequence at least 70% identical to
SEQ ID NO: 2 or 4; and (f) a nucleotide sequence that is the complement of any one of (a)-(f).
[0011] The isolated DLT polypeptides provided herein include (a) the amino acid sequence of SEQ ID NO: 2 or 4, (b) an amino acid sequence derived from SEQ ID NO: 2 or 4 by substitution and/or deletion and/or addition of one or more amino acid residues wherein the amino acid sequence is capable of altering tiller number and (c) an amino acid sequence at least
70% identical to SEQ ID NO: 2 or 4.
[0012] The host cells provided herein include those comprising the isolated polynucleotides and vectors of the present invention. The host cell can be from an animal, plant, or microorganism, such as E. coli. Plant cells are particularly contemplated. The host cell can be isolated, excised, or cultivated. The host cell may also be part of a plant.
[0013] The present invention further relates to a plant or a part of a plant that comprises a host cell of the present invention. Rice is particularly contemplated. The present invention also relates to the transgenic seeds of the plants.
[0014] The present invention further relates to a method for producing a plant comprising regenerating a transgenic plant from a host cell of the present invention, or hybridizing a transgenic plant of the present invention to another non-transgenic plant. Plants produced by these methods are also encompassed by the present invention, and rice is particularly contemplated.
[0015] The present invention further relates to methods of altering a trait in a plant or part of a plant using the isolated polynucleotides, polypeptides, constructs and vectors of the present invention. These traits include altering (i.e., increasing or decreasing) tiller number in comparison to a corresponding wild type plant and dwarf stature. In one embodiment, these traits are altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant.
[0016] The present invention further relates to the use of the isolated polynucleotides, polypeptides, constructs and vectors of the present invention to alter tiller number in a plant. In one embodiment, tiller number is altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant. BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS [0017] Figure 1 shows the phenotype of dlt mutant and wild type (WT) rice at (a) the vegetative phase and (b) the productive phase.
[0018] Figure 2(a) is a linkage map of DLT. DLTis located between S240 and S1551 on chromosome 6, near the 5' telomere. Sequence-tagged site markers are named according to their chromosome physical positions, and relevant recombinant numbers are indicated above the linkage map. PAC (Pl -derived artificial chromosome) or BAC clones in the dlt mutation candidate region are shown under the linkage map. Figure 2(b) shows the structure of the putative DL T full-length cDNA. The position of the mutation is shown. The underlined and italicized TGA is the putative new stop codon of an aberrant DLT in the dlt mutant. Arrows indicate the positions of the primer pairs used for RT-PCR of DLT. Figure 2(c) is an RT-PCR of DL T. The band sizes of DLT are indicated. ACTINl was used as a control. Figure 2(d) is a protein sequence alignment of DLT (OsGRAS32) and AtGRAS8 with AtGAI, AtRGAl, and AtSCR using Cluster W. The arrow indicates the mutation site. Five conserved motifs specific to GRAS proteins are indicated (leucine heptad I, VHIID, leucine heptad I, PFYRE, and SAW motifs). Conserved amino acids are highlighted in black and gray.
[0019] Figure 3 demonstrates the results of phenotypic complementation by introduction of the DLT gene into dlt mutants. Figures 3 (a) and 3(b) show the gross morphology at (a) the vegetative phase and (b) the productive phase of WT, transgenic (dlt-c) and mutant plants. Figure 3(c) is a PCR of WT, transgenic (dlt-c) and mutant plants. Arrows indicate band size. [0020] Figure 4(a) shows the lamina joint bending response to various amounts of 24-epiBL by the micro-drop method. Figure 4(b) shows the lamina joint bending response to 5 ng/ml 24- epiBL by the excised leaf segment method. Figure 4(c) shows the coleoptile elongation response to 0.1 μM 24-epiBL.
[0021] Figure 5(a) shows the expression of DLT in various organs analyzed by quantitative RT-PCR analysis. Panicles and culms were collected when they had reached 1 cm length. The SAM, root, and the third leaf sheath and blade were harvested from 2-week-old plants. Figure 5(b) shows the expression pattern of DLT in culm tissues. Intl to Int5 represent five internodes counted from top to bottom. The lowest zones of each internode were collected to extract RNA. [0022] Figure 6 shows the GUS staining of PRODLT :GUS transgenic line tissues. Pane 1 shows a longitudinal section after 3 days germination. The arrow indicates the SAM. Pane 2 shows a longitudinal section of 7-day-old seedling after germination in dark. A primary root is also shown. The arrow indicates the SAM. Pane 3 shows an unexpanded fourth leaf without greening from a 2-week old seedling. Pane 4 shows the microscopic observation of a cross- section of pane 3. Pane 5 shows a third leaf from a 2-week-old seedling. The arrow indicates the lamina joint. Pane 6 shows a longitudinal section of a young culm. The arrow indicates the shoot apex. Pane 7 shows a full-length elongating uppermost internode. Pane 8 shows a full- length elongated third internode. Pane 9 is a cross-section of the lowest part of pane 7. Pane 10 is amagnified image of part of pane 9. Pane 11 shows a young spikelet. Pane 12 shows an older floret. Pane 13 shows a young root with lateral root protruding. Pane 14 shows a lateral root. Scale bars = 1 mm (panes 1-3, 5, 6, 9, and 11-14), 100 μm (panes 4 and 10) or 1 cm ( panes 7 and 8).
[0023] Figure 7(a) is a quantitative RT-PCR analysis of transcription levels for DLT at various times after exogenous 1 μM 24-epiBL treatment. Figure 7(b) shows increased expression of DLT in d.2-1 and dll-2 mutants assayed by quantitative RT-PCR analysis. Shiokari plants are used as the WT control, and WT expression is set at 1.0. [0024] Figure 8 shows a comparison of brassinosteroid (BR)-related gene expression between WT and dlt mutants with or without 24-epiBL. Gene expression was normalized to that of the rice ACTINl gene, and levels in WT or levels without 24-epiBL treatment are set as 1.0. Figure 8(a) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT 'in WT and the dlt mutant. Figure 8(b) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT in WT and the dlt mutant grown on half-strength MS with or without 1 μM 24-epiBL. Figure 8(c) shows the quantitative RT-PCR analysis of expression of BR downstream genes and signaling genes.
[0025] Figure 9 shows an electrophoretic mobility shift assay. A WT DNA probe derived from the DLT promoter was incubated with 200 ng recombinant OsBZRl . Competition reactions with either unlabeled WT probe or the mutant (Mt) form were performed to demonstrate the specific binding of OsBZRl to the BRRE in the DLT promoter. [0026] Figure 10(a) shows the relative level of DLT expression in ten transgenic To plants. Figure 10(b) shows the differing phenotypes of wild type and transgenic T0 plants. [0027] Figure 11 is a statistical analysis of the tiller number often T2 transgenic plant lines performed during the heading stage. Plant line number is shown on the x-axis, while tiller number is shown on the y-axis. The mean tiller number and standard deviation are provided. Statistically significant differences generated from a Student's t-test are indicated at the P<0.05 (*) and P<0.01 (**) levels.
DETAILED DESCRIPTION OF THE INVENTION [0028] Nucleic Acids and Proteins
[0029] As used herein, the terms "nucleic acid", "polynucleotide", "polynucleotide molecule", "polynucleotide sequence" and plural variants are used interchangeably to refer to a wide variety of molecules, including single strand and double strand DNA and RNA molecules, cDNA sequences, genomic DNA sequences of exons and introns, chemically synthesized DNA and RNA sequences, and sense strands and corresponding antisense strands. Polynucleotides of the invention may also comprise known analogs of natural nucleotides that have similar properties as the reference natural nucleic acid.
[0030] As used herein, the terms "polypeptide", "protein" and plural variants are used interchangeably and refer to a compound made up of a single chain of amino acids joined by peptide bonds. Polypeptides of the invention may comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Polypeptides may include both L-form and D-form amino acids. [0031] Representative non-genetically encoded amino acids include but are not limited to 2- aminoadipic acid; 3-aminoadipic acid; β-aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2- aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N- ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N- methylvaline; norvaline; norleucine; and ornithine.
[0032] Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
[0033] Exemplary DLT polynucleotides of the invention are set forth as SEQ ID NOs: 1 and 2 and substantially identical sequences encoding proteins capable of altering the tiller number of a plant. Exemplary DLT polypeptides of the invention are set forth as SEQ ID NOs: 2 and 4 and substantially identical proteins capable of altering the tiller number of a plant. [0034] Substantially identical sequences are those that have at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In an especially preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acids or proteins perform substantially the same function. Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population. An allelic difference may be as small as one base pair. Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations. A mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues. Substantially identical nucleic acids are also identified as nucleic acids that hybridize specifically to or hybridize substantially to a reference sequence (e.g., SEQ ID NO: 1). [0035] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. [0036] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol, 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI), or by visual inspection (see Ausubel et al., infra).
[0037] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. MoI. Biol, 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989)).
[0038] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[0039] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. Stringent conditions are those under which a nucleic acid probe will typically hybridize to its target sequence but to no other sequences when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA). Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments such as Southern and Northern blot analyses are both sequence- and environment-dependent. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I chapter 2, Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5 0C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
[0040] The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42 0C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72 0C for about 15 minutes. Another example of stringent wash conditions is a 0.2X SSC wash at 65 0C for 15 minutes {see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is IX SSC at 45 0C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4X - 6X SSC at 40 0C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M sodium ions, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 0C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
[0041] The following are examples of hybridization and wash conditions that may be used to identify nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention. A substantially identical nucleotide sequence preferably hybridizes to a reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 0C with washing in 2X SSC, 0.1% SDS at 50 0C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 0C with washing in IX SSC, 0.1% SDS at 50 0C, still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 0C with washing in 0.5X SSC, 0.1% SDS at 50 0C, even more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 0C with washing in 0.1X SSC, 0.1% SDS at 50 0C, and most preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 0C with washing in 0.1X SSC, 0.1% SDS at 65 0C.
[0042] A further indication that two nucleic acid sequences or proteins are substantially identical is that the that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents, or are immunologically cross-reactive with, or specifically bind to, each other. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This may occur, for example, when two nucleotide sequences comprise conservatively substituted variants as permitted by the genetic code. This also includes degenerate codon substitutions wherein the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acids Res., 19:5081(1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); and Rossolini et al. MoI. Cell Probes, 8:91-98 (1994)). However, both the polynucleotides and the polypeptides of the present invention may be conservatively substituted at one or more residues. Examples of conservative amino acid substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. [0043] Nucleic acids of the invention also comprise nucleic acids complementary to SEQ ID NOs: 1 and 3, and subsequences and elongated sequences of SEQ ID NOs: 1 and 3 and complementary sequences thereof. Complementary sequences are two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs. Like other polynucleotides in accordance with the present invention, complementary sequences maybe substantially similar to one another as described previously. A particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
[0044] A subsequence is a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence. An exemplary subsequence is a probe or a primer. An elongated sequence is one in which nucleotides (or other analogous molecules) are added to a nucleic acid sequence. For example, a polymerase (e.g., a DNA polymerase) may add sequences at the 3' terminus of the nucleic acid molecule. In addition, the nucleotide sequence may be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, introns, additional restriction enzyme sites, multiple cloning sites, and other coding segments. Thus, the present invention also provides vectors comprising the disclosed nucleic acids, including vectors for recombinant expression, wherein a nucleic acid of the invention is operatively linked to a functional promoter. When operatively linked to a nucleic acid, a promoter is in functional combination with the nucleic acid such that the transcription of the nucleic acid is controlled and regulated by the promoter region. Vectors refer to nucleic acids capable of replication in a host cell, such as plasmids, cosmids, and viral vectors.
[0045] Polynucleotides of the present invention may be cloned, synthesized, altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art. Site-specific mutagenesis to create base pair changes, deletions, or small insertions is also known in the art (see e.g., Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual. 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Silhavy et al., Experiments with Gene Fusions, 1984, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover & Hames, DNA Cloning: A Practical Approach, 2nd ed., 1995, IRL Press at Oxford University Press, Oxford/New York; Ausubel (ed.) Short Protocols in Molecular Biology, 3rd ed., 1995, Wiley, New York).
[0046] Isolated polypeptides of the invention may be purified and characterized using a variety of standard techniques that are known to the skilled artisan (see e.g., Schroder et al., The Peptides, 1965, Academic Press, New York; Bodanszky, Principles of Peptide Synthesis, 2nd rev. ed. 1993, Springer- Verlag, Berlin/ New York; Ausubel (ed.), Short Protocols in Molecular Biology. 3rd ed., 1995, Wiley, New York).
[0047] The present invention also encompasses methods for detecting a nucleic acid molecule that encodes a DLT protein. Such methods may be used to detect gene variants or altered gene expression. Sequences detected by methods of the invention may detected, subcloned, sequenced, and further evaluated by any measure well known in the art using any method usually applied to the detection of a specific DNA sequence. Thus, the nucleic acids of the present invention may be used to clone genes and genomic DNA comprising the disclosed sequences. Alternatively, the nucleic acids of the present invention may be used to clone genes and genomic DNA of related sequences. Levels of a DL T nucleic acid molecule may be measured, for example, using an RT-PCR assay (see e.g., Chiang, J Chromatogr. A., 806:209- 218 (1998) and references cited therein).
[0048] The present invention also encompasses genetic assays using DLT nucleic acids for quantitative trait loci (QTL) analysis and to screen for genetic variants, for example by allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sd. USA, 80(l):278-282 (1983)), oligonucleotide ligation assays (OLAs) (Nickerson et al., Proc. Natl. Acad. ScL USA, 87(22):8923-8927 (1990)), single-strand conformation polymorphism (SSCP) analysis (Orita et al., Proc. Natl. Acad. Sci. USA, 86(8):2766-2770 (1989)), SSCP/heteroduplex analysis, enzyme mismatch cleavage, direct sequence analysis of amplified exons (Kestila et al., MoI. Cell, l(4):575-582 (1998); Yuan et al., Hum. Mutat., 14(5):440-446 (1999)), allele-specific hybridization (Stoneking et al., Am. J. Hum. Genet., 48(2):370-382 (1991)), and restriction analysis of amplified genomic DNA containing the specific mutation. Automated methods may also be applied to large-scale characterization of single nucleotide polymorphisms (Wang et al., Am. J. Physiol, 1998, 274(4 Pt 2):H1132-1140 (1992); Brookes, Gene, 234(2): 177-186 (1999)). Preferred detection methods are non-electrophoretic, including, for example, the TAQMAN™ allelic discrimination assay, PCR-OLA, molecular beacons, padlock probes, and well fluorescence (see Landegren et al., Genome Res., 8:769-776 (1998) and references cited therein). [0049] The present invention also encompasses functional fragments of a DLT polypeptide, for example, fragments that have the ability to alter tiller number similar to that of SEQ ID NOs: 2 and 4. Functional polypeptide sequences that are longer than the disclosed sequences are also encompassed. For example, one or more amino acids may be added to the N-terminus or C- terminus of an antibody polypeptide. Such additional amino acids may be employed in a variety of applications, including but not limited to purification applications. Methods of preparing elongated proteins are known in the art.
[0050] The present invention also encompasses methods for detecting a polypeptide. Such methods can be used, for example, to determine levels of protein expression and correlate the level of expression with the presence or change in phenotype, trait, or level of expression in a different gene or gene product. In certain embodiments, the method involves an immunochemical reaction with an antibody that specifically recognizes a protein. Techniques for detecting such antibody-antigen conjugates or complexes are known in the art and include but are not limited to centrifugation, affinity chromatography and other immunochemical methods (see e.g., Ishikawa, Ultrasensitive and Rapid Enzyme Immunoassay, 1999, Elsevier, Amsterdam/New York, United States of America; Law, Immunoassay: A Practical Guide, 1996, Taylor & Francis, London/Bristol, Pennsylvania, United States of America; Liddell et al., Antibody Technology. 1995, Bios Scientific Publishers, Oxford, United Kingdom; and references cited therein).
[0051] DLT Expression Systems
[0052] An expression system refers to a host cell comprising a heterologous nucleic acid and the protein encoded by the heterologous nucleic acid. For example, a heterologous expression system may comprise a host cell transfected with a construct comprising a DLT nucleic acid encoding a protein operatively linked to a promoter, or a cell line produced by introduction of DLT nucleic acids into a host cell genome. The expression system may further comprise one or more additional heterologous nucleic acids relevant to DLT function, such as targets of DLT transcriptional activation or repression activity. These additional nucleic acids may be expressed as a single construct or multiple constructs. [0053] A construct for expressing a DLT protein may include a vector sequence and a DLT nucleotide sequence, wherein the DL T nucleotide sequence is operatively linked to a promoter sequence. A construct for recombinant DL T expression may also comprise transcription termination signals and sequences required for proper translation of the nucleotide sequence. Preparation of an expression construct, including addition of translation and termination signal sequences, is known to one skilled in the art.
[0054] The promoter may be any polynucleotide sequence which shows transcriptional activity in the chosen plant cells, plant parts, or plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Where the promoter is native or endogenous to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence of the invention, the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention. The promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic. Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley et al., Nucleic Acids Res., 15:2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized {see e.g., Roberts et al., Proc. Natl. Acad. Sd. USA, 76:760-4 (1979)). Many suitable promoters for use in plants are well known in the art. [0055] For example, suitable constitutive promoters for use in plants include the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PClSV) promoter (U.S. Patent No. 5,850,019); the 35S and 19S promoters from cauliflower mosaic virus (CaMV) (Odell et al., Nature, 313:810-812 (1985) and U.S. Patent No. 5,352,605); the promoters of Chlorella virus methyltransferase genes (U.S. Patent No. 5,563,328) and the full-length transcript promoter from figwort mosaic virus (FMV) (U.S. Patent No. 5,378,619); the promoters from such genes as rice actin (McElroy et al., Plant Cell, 2:163-171 (1990)); ubiquitin (Binet et al., Plant Science, 79:87-94 (1991)), maize (Christensen et al., Plant Molec. Biol, 12:619-632 (1989)), and arabidopsis (Norris et al., Plant Molec. Biol, 21:895-906 (1993); Christensen et al., Plant MoI Biol, 18:675-689 (1982)); pEMU (Last et al., Theor. Appl Genet., 81:581-588 (1991)); MAS (Velten et al., EMBOJ., 3:2723-2730 (1984)); maize H3 histone (Lepetit et al., MoI Gen. Genet., 1992, 231:276-285 (1992); Atanassova et al., Plant J., 2(3):291-300 (1992)); Brassica napus ALS3 (PCT International Publication No. WO 97/41228); and promoters of various Agrobacterium genes (e.g., U.S. Patent Nos. 4,771,002; 5,102,796; 5,182,200; and 5,428,147). [0056] Suitable inducible promoters for use in plants include the promoter from the ACEl system which responds to copper (Mett et al., Proc. Natl. Acad. ScL USA, 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., MoI. Gen. Genetics, 227:229-237 (1991); and Gatz et al., MoI. Gen. Genetics, 243:32-38 (1994)); and the promoter of the Tet repressor from TnIO (Gatz et al., MoI. Gen. Genet. , 227:229-237 (1991)). Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. ScL USA, 88:10421 (1991)) or the recent application of a chimeric transcription activator, XVE, for use in an estrogen receptor-based inducible plant expression system activated by estradiol (Zuo et al., Plant J., 24:265-273 (2000)). Other inducible promoters for use in plants are described in EP 332104, PCT International Publication Nos. WO 93/21334 and WO 97/06269. Promoters composed of portions of other promoters and partially or totally synthetic promoters can also be used {see e.g., Ni et al., Plant J., 7:661-676 (1995) and PCT International Publication No. WO 95/14098 describing such promoters for use in plants).
[0057] Tissue-specific or tissue-preferential promoters useful for the expression of the novel DLT genes of the invention in plants, including the cotton rubisco promoter disclosed in U.S. Patent No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent No. 5,604,121; and the cestrum yellow leaf curling virus promoter disclosed in PCT International Publication No. WO 01/73087. Chemically inducible promoters useful for directing the expression of DLT genes in plants are disclosed in U.S. Patent No. 5,614,395.
[0058] The promoter may include, or be modified to include, one or more enhancer elements to thereby provide for higher levels of transcription. Suitable enhancer elements for use in plants include the PClSV enhancer element (U.S. Patent No. 5,850,019), the CaMV 35S enhancer element (U.S. Patent Nos. 5,106,739 and 5,164,316) and the FMV enhancer element (Maiti et al., Transgenic Res., 6:143-156 (1997)). See also PCT International Publication No. WO 96/23898. [0059] Such constructs can contain a 'signal sequence' or 'leader sequence' to facilitate co- translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted. For example, the construct can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. A signal sequence is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. A leader sequence refers to any sequence that, when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Plant expression cassettes may also contain an intron, such that mRNA processing of the intron is required for expression.
[0060] Such constructs can also contain 5 ' and 3 ' untranslated regions. A 3 ' untranslated region is a polynucleotide located downstream of a coding sequence. Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions. A 5' untranslated region is a polynucleotide located upstream of a coding sequence. [0061] The termination region may be native with the transcriptional initiation region, may be native with the sequence of the present invention, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions (see e.g., Guerineau et al., MoI. Gen. Genet., 262:141-144 (1991); Proudfoot, Cell, 64:671-674 (1991); Sanfacon et al., Genes Dev., 5:141-149 (1991); Mogen et al., Plant Cell, 2:1261-1272 (1990); Munroe et al., Gene, 91:151-158 (1990); Ballas et al., Nucleic Acids Res., 17:7891-7903 (1989); and Joshi et al., Nucleic Acid Res., 15:9627-9639 (1987)).
[0062] Where appropriate, the vector and DLT sequences may be optimized for increased expression in the transformed host cell. That is, the sequences can be synthesized using host cell-preferred codons for improving expression, or may be synthesized using codons at a host- preferred codon usage frequency. Generally, the GC content of the polynucleotide will be increased {see e.g., Campbell et al., Plant Physiol, 92:1-11 (1990) for a discussion of host- preferred codon usage). Methods are known in the art for synthesizing host-preferred polynucleotides {see e.g., U.S. Patent Nos. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Application Publication Nos. 20040005600 and 20010003849, and Murray et al., Nucleic Acids Res., 17:477-498 (1989).
[0063] In certain embodiments, polynucleotides of interest are targeted to the chloroplast for expression. In this manner, where the polynucleotide of interest is not directly inserted into the chloroplast, the expression cassette may additionally contain a polynucleotide encoding a transit peptide to direct the nucleotide of interest to the chloroplasts. Such transit peptides are known in the art (see e.g., Von Heijne et al., Plant MoI. Biol. Rep., 9:104-126 (1991); Clark et al., J. Biol. Chem., 264:17544-17550 (1989); Della-Cioppa et al., Plant Physiol, 84:965-968 (1987); Romer et al., Biochem. Biophys. Res. Commun., 196:1414-1421 (1993); and Shah et al., Science, 233:478-481 (1986)). The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons (see e.g., U.S. Patent No. 5,380,831). [0064] A plant expression cassette (i.e., a DLT open reading frame operatively linked to a promoter) can be inserted into a plant transformation vector, which allows for the transformation of DNA into a cell. Such a molecule may consist of one or more expression cassettes, and may be organized into more than one vector DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens et al., Trends in Plant Science, 5:446-451 (2000)).
[0065] A plant transformation vector comprises one or more DNA vectors for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that comprise more than one contiguous DNA segment. These vectors are often referred to in the art as binary vectors. Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a polynucleotide of interest (i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). [0066] For certain target species, different antibiotic or herbicide selectable markers may be preferred. Selection markers used routinely in transformation include the nptllgene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, Gene, 19:259-268 (1982); and Bevan et al., Nature, 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res., 18:1062 (1990), and Spencer et al., Theor. Appl. Genet., 79:625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, MoI. Cell. Biol, 4:2929-2931 (1984)), the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J., 2(7):1099-l 104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629).
[0067] Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker sequence and the sequence of interest are located between the left and right borders. Often a second plasmid vector contains the transacting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as in understood in the art (Hellens et al., 2000). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHAlOl, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for introduction of polynucleotides into plants by other methods such as, e.g., microprojection, microinjection, electroporation, and polyethylene glycol. [0068] In another embodiment, a nucleotide sequence of the present invention is directly transformed into a plastid genome. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT International Application Publication WO 95/16783, and in McBride et al., Proc. Natl. Acad. Sci. USA, 91:7301-7305 (1994). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526-8530 (1990); Staub et al., Plant Cell, 4:39-45 (1992)). This results in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al., EMBOJ., 12:601-606 (1993)). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab et al., Proc. Natl. Acad. Sci. USA, 90:913-917 (1993)). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, Nucl. Acids Res., 19:4083-4089 (1991)). Other selectable markers useful for plastid transformation are known in the art. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleotide sequence of the present invention is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
[0069] Host Cells
[0070] Host cells are cells into which a heterologous nucleic acid molecule of the invention may be introduced. Representative eukaryotic host cells include yeast and plant cells, as well as prokaryotic hosts such as E.coli and B. subtilis. Preferred host cells for functional assays substantially or completely lack endogenous expression of a DLT protein. [0071] A host cell strain may be chosen which modulates the expression of the recombinant sequence, or modifies and processes the gene product in a specific manner. For example, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host cells may be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system may be used to produce a non-glycosylated core protein product, and expression in yeast will produce a glycosylated product.
[0072] The present invention further encompasses recombinant expression of a DLT protein in a stable cell line. Methods for generating a stable cell line following transformation of a heterologous construct into a host cell are known in the art {see e.g., Joyner, Gene Targeting: A Practical Approach. 1993, Oxford University Press, Oxford/New York). Thus, transformed cells, tissues, and plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny or propagated forms thereof.
[0073] DLT Knockout Plants
[0074] The present invention also provides DLT knockout plants comprising a disruption of a
DLT\ocMS. A disrupted gene may result in expression of an altered level of full-length DLT protein or expression of a mutated variant DLT protein (e.g., SEQ ID NO: 4). Plants with complete or partial functional inactivation of the DLT gene may be generated, e.g., by expressing a mutant DL T allele in the plant.
[0075] A knockout plant in accordance with the present invention may also be prepared using anti-sense, double-stranded RNA, or ribozyme DL T constructs, driven by a universal or tissue-specific promoter to reduce levels of DLT gene expression in somatic cells, thus achieving a "knock-down" phenotype. The present invention also provides the generation of plants with conditional or inducible inactivation of DLT.
[0076] The present invention also encompasses transgenic plants with specific "knocked-in" modifications in the disclosed DLT gene, for example to create an over-expression mutant having a dominant negative phenotype. Thus, "knocked-in" modifications include the expression of mutant alleles of the DLT gene.
[0077] DL T knockout plants may be prepared in monocot or dicot plants, such as maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees. Rice is particularly contemplated. As used herein, a plant refers to a whole plant, a plant organ (e.g., root, stem, leaf, flower bud, or embryo), a seed, a plant cell, a propagule, an embryo, other plant parts (e.g., protoplasts, pollen, pollen tubes, ovules, embryo sacs, zygotes) and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
[0078] For preparation of a DL T knockout plant, introduction of a polynucleotide into plant cells is accomplished by one of several techniques known in the art, including but not limited to electroporation or chemical transformation {see e.g., Ausubel, ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Indiana). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test polynucleotide sequence) from non-transformed cells (those not containing or not expressing the test polynucleotide sequence). In one aspect of the invention, genes are useful as a marker to assess introduction of DNA into plant cells. Transgenic plants, transformed plants, or stably transformed plants, or cells, tissues or seed of any of the foregoing, refer to plants that have incorporated or integrated exogenous polynucleotides into the plant cell. Stable transformation refers to introduction of a polynucleotide construct into a plant such that it integrates into the genome of the plant and is capable of being inherited by progeny thereof. [0079] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (i.e., temperature and/or herbicide). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (see e.g., Hiei et al., Plant J., 6:271-282 (1994); and Ishida et al., Nat. Biotechnol, 14:745-750 (1996)). A general description of the techniques and methods for generating transgenic plants are found in Ayres et al., CRC Crit. Rev. Plant ScL, 13:219-239 (1994); and Bommineni et al., Maydica, 42:107-120 (1997). Since the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants. Subsequently, molecular and biochemical methods can be used for confirming the presence of the integrated nucleotide(s) of interest in the genome of transgenic plant.
[0080] Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods to transfer DNA (see e.g., Hiei et al., Plant J., 6:271-282 (1994); Ishida et al., Nat. Biotechnol., 14:745-750 (1996); Ayres et al., CRC Crit. Rev. Plant ScL, 13:219-239 (1994); and Bommineni et al., Maydica, 1997, 42:107-120 (1997)).
[0081] There are three common methods for transforming plant cells with Agrobacterium. The first method is co-cultivation of Agrobacterium with cultured isolated protoplasts. This method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts. The second method is transformation of cells or tissues with Agrobacterium. This method requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants. The third method is transformation of seeds, apices or meristems with Agrobacterium. This method requires micropropagation. [0082] The efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens (Shahla et al., Plant Molec. Biol, 8:291-298 (1987)). Alternatively, transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles {see e.g., Bidney et al., Plant Molec. Biol, 18:301-313 (1992).
[0083] In one embodiment, the plant cells are transfected with vectors via particle bombardment (i.e., with a gene gun). Particle mediated gene transfer methods are known in the art, are commercially available, and include, but are not limited to, the gas driven gene delivery instrument described in U.S. Patent No. 5,584,807. This method involves coating the polynucleotide sequence of interest onto heavy metal particles, and accelerating the coated particles under the pressure of compressed gas for delivery to the target tissue. [0084] Other particle bombardment methods are also available for the introduction of heterologous polynucleotide sequences into plant cells. Generally, these methods involve depositing the polynucleotide sequence of interest upon the surface of small, dense particles of a material such as gold, platinum, or tungsten. The coated particles are themselves then coated onto either a rigid surface, such as a metal plate, or onto a carrier sheet made of a fragile material such as mylar. The coated sheet is then accelerated toward the target biological tissue. The use of the flat sheet generates a uniform spread of accelerated particles that maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the polynucleotide sample into the target tissue.
[0085] Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide of interest, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers that are appropriate for the particular cell system that is used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ., 20:125 (1994)).
[0086] The cells that have been transformed may be grown into plants in accordance with conventional ways {see e.g., McCormick et al., Plant Cell Rep., 5:81-84 (1986)). These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as transgenic seed) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
[0087] Transgenic plants of the invention can be homozygous for the added polynucleotides; i.e., a transgenic plant that contains two added sequences, one sequence at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains the added sequences according to the invention, germinating some of the seed produced and analyzing the resulting plants produced for enhanced enzyme activity (i.e., herbicide resistance) and/or increased plant yield relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
[0088] It is to be understood that two different transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous polynucleotides. Selfing of appropriate progeny can produce plants that are homozygous for all added, exogenous polynucleotides that encode a polypeptide of the present invention. Back-crossing to a parental plant and outcrossing with a non-transgenic plant are also contemplated. [0089] Following introduction of DNA into plant cells, the transformation or integration of the polynucleotide into the plant genome is confirmed by various methods such as analysis of polynucleotides, polypeptides and metabolites associated with the integrated sequence. [0090] DLT Inhibitors
[0091] The present invention further discloses assays to identify DLT binding partners and DLT inhibitors. DLT antagonists/inhibitors are agents that alter chemical and biological activities or properties of a DLT protein. Methods of identifying inhibitors involve assaying a reduced level or quality of DLT function in the presence of one or more agents. Exemplary DLT inhibitors include small molecules as well as biological inhibitors as described herein below. [0092] As used herein, the term "agent" refers to any substance that potentially interacts with a DLT nucleic acid or protein, including any of synthetic, recombinant, or natural origin. An agent suspected to interact with a protein may be evaluated for such an interaction using the methods disclosed herein.
[0093] Exemplary agents include but are not limited to peptides, proteins, nucleic acids, small molecules (e.g., chemical compounds), antibodies or fragments thereof, nucleic acid- protein fusions, any other affinity agent, and combinations thereof. An agent to be tested may be a purified molecule, a homogenous sample, or a mixture of molecules or compounds. [0094] A small molecule refers to a compound, for example an organic compound, with a molecular weight of less than about 1,000 daltons, more preferably less than about 750 daltons, still more preferably less than about 600 daltons, and still more preferably less than about 500 daltons. A small molecule also preferably has a computed log octanol-water partition coefficient in the range of about -A to about +14, more preferably in the range of about -2 to about +7.5. [0095] Exemplary nucleic acids that may be used to disrupt DLT function include antisense RNA and small interfering RNAs (siRNAs) {see e.g., U.S. Application Publication No. 20060095987). These inhibitory molecules may be prepared based upon the DLT gene sequence and known features of inhibitory nucleic acids {see e.g., Van der Krol et al., Plant Cell, 2:291- 299 (1990); Napoli et al., Plant Cell, 2:279-289 (1990); English et al., Plant Cell, 8:179-188 (1996); and Waterhouse et al., Nature Rev. Genet, 2003, 4:29-38 (2003). [0096] Agents may be obtained or prepared as a library or collection of molecules. A library may contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more. A molecule may comprise a naturally occurring molecule, a recombinant molecule, or a synthetic molecule. A plurality of agents in a library may be assayed simultaneously. Optionally, agents derived from different libraries may be pooled for simultaneous evaluation. [0097] Representative libraries include but are not limited to a peptide library (U.S. Patent Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Patent Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Patent Nos. 7,338,762; 7,329,742; 6,949,379; 6,180,348; and 5,756,291), a small molecule library (U.S. Patent Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (U.S. Patent Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667988), a library of nucleic acid-protein fusions (U.S. Patent No. 6,214,553), and a library of any other affinity agent that may potentially bind to a DLT protein.
[0098] A library may comprise a random collection of molecules. Alternatively, a library may comprise a collection of molecules having a bias for a particular sequence, structure, or conformation, for example, as for inhibitory nucleic acids (see e.g., U.S. Patent Nos. 5,264,563 and 5,824,483). Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. patents cited herein above. Numerous libraries are also commercially available.
[0099] A control level or quality of DLT activity refers to a level or quality of wild type DLT activity, for example, when using a recombinant expression system comprising expression of SEQ ID NOs: 1 or 3. When evaluating the inhibiting capacity of an agent, a control level or quality of DLT activity comprises a level or quality of activity in the absence of the agent. A control level may also be established by a phenotype or other measureable trait. [00100] Methods of identifying DLT inhibitors also require that the inhibiting capacity of an agent be assayed. Assaying the inhibiting capacity of an agent may comprise determining a level of DLT gene expression; determining DNA binding activity of a recombinantly expressed DLT protein; determining an active conformation of a DLT protein; or determining a change in a trait in response to binding of a DLT inhibitor (e.g., increased or decreased tiller number). In particular embodiments, a method of identifying a DLT inhibitor may comprise (a) providing a cell, plant, or plant part expressing a DLT protein; (b) contacting the cell, plant, or plant part with an agent; (c) examining the cell, plant, or plant part for a change in a trait as compared to a control; and (d) selecting an agent that induces a change in the trait as compared to a control. Any of the agents so identified in the disclosed inhibitory or binding assays (see hereinafter) may be subsequently applied to a cell, plant or plant part as desired to effectuate a change in that cell, plant or plant part. [00101] The present invention also encompasses a rapid and high throughput screening method that relies on the methods described herein. This screening method comprises separately contacting a DLT protein with a plurality of agents. In such a screening method the plurality of agents may comprise more than about 104 samples, or more than about 105 samples, or more than about 106 samples.
[00102] The in vitro and cellular assays of the invention may comprise soluble assays, or may further comprise a solid phase substrate for immobilizing one or more components of the assay. For example, a DLT protein, or a cell expressing a DLT protein, may be bound directly to a solid state component via a covalent or non-covalent linkage. Optionally, the binding may include a linker molecule or tag that mediates indirect binding of a DLT protein to a substrate.
[00103] DLT Binding Assays
[00104] The present invention also encompasses methods of identifying of a DLT inhibitor by determining specific binding of a substance (e.g., an agent described previously) to a DLT protein. For example, a method of identifying a DLT binding partner may comprise: (a) providing a DLT protein of SEQ ID NO: 2 or 4; (b) contacting the DLT protein with one or more agents under conditions sufficient for binding; (c) assaying binding of the agent to the isolated DLT protein; and (d) selecting an agent that demonstrates specific binding to the DLT protein. Specific binding may also encompass a quality or state of mutual action such that binding of an agent to a DLT protein is inhibitory.
[00105] Specific binding refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials. The binding of an agent to a DLT protein may be considered specific if the binding affinity is about IxIO4M"1 to about 1x106 M"1 or greater. Specific binding also refers to saturable binding. To demonstrate saturable binding of an agent to a DLT protein, Scatchard analysis may be carried out as described, for example, by Mak et al, J Biol. Chem., 264:21613-21618 (1989). [00106] Several techniques may be used to detect interactions between a DLT protein and an agent without employing a known competitive inhibitor. Representative methods include, but are not limited to, Fluorescence Correlation Spectroscopy, Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Spectroscopy, and BIACORE® technology, each technique described herein below. These methods are amenable to automated, high-throughput screening.
[00107] Fluorescence Correlation Spectroscopy (FCS) measures the average diffusion rate of a fluorescent molecule within a small sample volume. The sample size may be as low as 103 fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium. The diffusion rate is a function of the mass of the molecule and decreases as the mass increases. FCS may therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding. In a typical experiment, the target to be analyzed (e.g., a DLT protein) is expressed as a recombinant protein with a sequence tag, such as a poly-histidine sequence, inserted at the N-terminus or C-terminus. The expression is mediated in a host cell, such as E.coli, yeast, Xenopus oocytes, or mammalian cells. The protein is purified using chromatographic methods. For example, the poly-histidine tag may be used to bind the expressed protein to a metal chelate column such as Ni2+ chelated on iminodiacetic acid agarose. The protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPY™ reagent (available from Molecular Probes of Eugene, Oregon). The protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood of New York, New York). Ligand binding is determined by changes in the diffusion rate of the protein.
[00108] Surface-Enhanced Laser Desorption/Ionization (SELDI) was developed by Hutchens & Yip, Rapid Commun. Mass Spectrom., 1993, 7:576-580. When coupled to a time-of-flight mass spectrometer (TOF), SELDI provides a technique to rapidly analyze molecules retained on a chip. It may be applied to ligand-protein interaction analysis by covalently binding the target protein, or portion thereof, on the chip and analyzing by mass spectrometry the small molecules that bind to this protein (Worrall et al, Anal Chem., 1998, 70(4):750-756 (1998)). In a typical experiment, a target protein (e.g., a DLT protein) is recombinantly expressed and purified. The target protein is bound to a SELDI chip either by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. A chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler). The chip is then washed in solutions of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF. Ligands that specifically bind a target protein are identified by the stringency of the wash needed to elute them.
[00109] BIACORE® relies on changes in the refractive index at the surface layer upon binding of a ligand to a target protein (e.g., a DLT protein) immobilized on the layer. In this system, a collection of small ligands is injected sequentially in a 2-5 microliter cell, wherein the target protein is immobilized within the cell. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface. In general, the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein. In a typical experiment, a target protein is recombinantly expressed, purified, and bound to a BIACORE® chip. Binding may be facilitated by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction. A chip thus prepared is then exposed to one or more potential ligands via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler). The SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics of on rate and off rate allows the discrimination between non-specific and specific interaction (see also Homola et al., Sensors and Actuators, 54:3-15 (1999) and references therein).
[00110] Conformational Assays
[00111] The present invention also encompasses methods of identifying DLT binding partners and inhibitors that rely on a conformational change of a DLT protein when bound by or otherwise interacting with a substance (e.g., an agent described previously). For example, application of circular dichroism to solutions of macromolecules reveals the conformational states of these macromolecules. The technique may distinguish random coil, alpha helix, and beta chain conformational states.
[00112] To identify inhibitors of a DLT protein, circular dichroism analysis may be performed using a recombinantly expressed DLT protein. A DLT protein is purified, for example by ion exchange and size exclusion chromatography, and mixed with an agent. The mixture is subjected to circular dichroism. The conformation of a DLT protein in the presence of an agent is compared to a conformation of a DLT protein in the absence of the agent. A change in conformational state of a DLT protein in the presence of an agent identifies a DLT binding partner or inhibitor. Representative methods are described in U.S. Patent Nos. 5,776,859 and 5,780,242. Antagonistic activity of the inhibitor may be assessed using functional assays, such assaying nitrate content, nitrate uptake, lateral root growth, or plant biomass, as described herein. [00113] In accordance with the disclosed methods, cells expressing DLT may be provided in the form of a kit useful for performing an assay of DLT function. For example, a kit for detecting a DLT may include cells transfected with DNA encoding a full-length DLT protein and a medium for growing the cells.
[00114] Assays of DLT activity that employ transiently transfected cells may include a marker that distinguishes transfected cells from non-transfected cells. A marker may be encoded by or otherwise associated with a construct for DLT expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding DLT and the marker. Representative detectable molecules that are useful as markers include but are not limited to a heterologous nucleic acid, a protein encoded by a transfected construct (e.g., an enzyme or a fluorescent protein), a binding protein, and an antigen.
[00115] Assays employing cells expressing recombinant DLT or plants expressing DLT may additionally employ control cells or plants that are substantially devoid of native DLT and, optionally, proteins substantially similar to a DLT protein. When using transiently transfected cells, a control cell may comprise, for example, an untransfected host cell. When using a stable cell line expressing a DLT protein, a control cell may comprise, for example, a parent cell line used to derive the -DZr-expressing cell line.
[00116] Anti-DLT Antibodies
[00117] In another aspect of the invention, a method is provided for producing an antibody that specifically binds a DLT protein. According to the method, a full-length recombinant DLT protein is formulated so that it may be used as an effective immunogen, and used to immunize an animal so as to generate an immune response in the animal. The immune response is characterized by the production of antibodies that may be collected from the blood serum of the animal.
[00118] An antibody is an immunoglobulin protein, or antibody fragments that comprise an antigen binding site (e.g., Fab, modified Fab, Fab', F(ab')2 or Fv fragments, or a protein having at least one immunoglobulin light chain variable region or at least one immunoglobulin heavy chain region). Antibodies of the invention include diabodies, tetrameric antibodies, single chain antibodies, tretravalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and domain-specific antibodies that recognize a particular epitope. Cell lines that produce anti-DLT antibodies are also encompassed by the invention.
[00119] Specific binding of an antibody to a DLT protein refers to preferential binding to a DLT protein in a heterogeneous sample comprising multiple different antigens. Substantially lacking binding describes binding of an antibody to a control protein or sample, i.e., a level of binding characterized as non-specific or background binding. The binding of an antibody to an antigen is specific if the binding affinity is at least about 10~7 M or higher, such as at least about 10~8 M or higher, including at least about 10~9 M or higher, at least about 10"11 M or higher, or at least about 10~12 M or higher.
[00120] DLT antibodies prepared as disclosed herein may be used in methods known in the art relating to the expression and activity of DLT proteins, e.g., for cloning of nucleic acids encoding a DLT protein, immunopurification of a DLT protein, and detecting a DLT protein in a plant sample, and measuring levels of a DLT protein in plant samples. To perform such methods, an antibody of the present invention may further comprise a detectable label, including but not limited to a radioactive label, a fluorescent label, an epitope label, and a label that may be detected in vivo. Methods for selection of a label suitable for a particular detection technique, and methods for conjugating to or otherwise associating a detectable label with an antibody are known to one skilled in the art.
[00121] Examples
[00122] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teachings provided herein.
[00123] Example 1
[00124] Phenotype comparison [00125] A dwarf and low tillering mutant (dlt) was identified from a library of rice T-DNA insertion mutants from the (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). As shown in Figure 1, the dlt mutant is 60% as tall and has only half the number of tillers of the corresponding Zhonghua 11 wild-type (WT) plants. In addition, the mutant plant has a compact morphology, erect leaves and panicles, reduced fertility, and a reduced ratio of the second internode to the total internode length. Microscopic observation also revealed that the dlt mutant had decreased cell length and less organized cellular morphology. These observations are consistent with plants that exhibit altered brassinosteroid signaling and/or synthesis.
[00126] Example 2
[00127] Identification of the DLT gene
[00128] Genetic analysis suggested that the T-DNA insertion did not co-segregate with the mutant phenotype. The dlt mutant was crossed with Minghui 63, an indica variety, and F1 progeny had a WT phenotype. F2 progeny showed a 3:1 ratio between normal and mutant individuals, suggesting that the dlt mutation was caused by a single recessive gene. To clone the DLT gene, a mapping population with more than 3000 mutant individuals was constructed. Using only 300 individuals, the DLT gene was located to chromosome 6 between two sequence- tagged site markers, S240 and S 1551. Two recombinants were found with S 1551, and 39 recombinants were identified with S240 suggesting that the DLT gene is approximately 0.67 cM away from the S 1551 marker (see Figure 2(a)). As it was not possible to develop additional markers in this region due to low polymorphism between the japonica and indica varieties, the distance was estimated to be 134 kb away from S 1551.
[00129] Further analysis of this region led to to the identification of a putative gene approximately 105 kb away from S 1551, which encodes a GRAS family member. This putative gene, designated DZJ(SEQ ID NO: 18), has no intron, has a length of 3084 bp, with a 770 bp 5'-UTR, 1854 bp coding sequence and 460 bp 3'-UTR (see Figure 2(b)). DLT encodes a protein of 617 amino acids (DLT; SEQ ID NO: 2) that belongs to the plant-specific GRAS family. Alignment of DLT with Arabidopsis GAI, RGAl and SCR shows that DLT has the common structure of GRAS family proteins, with a variable C-terminus and a conserved N-terminus with five motifs: leucine heptad I, VHIID, leucine heptad II, PFYRE and SAW [Figure 2(d)]. [00130] Sequencing analysis of dlt (SEQ ID NO: 3) revealed a 62 bp deletion in the coding region of DLT in the mutant plant (see Figure 2(b)). The 62 bp deletion starts 524 bp from the initiation codon. The frameshift caused by the deletion generates an unrelated peptide (dlt; SEQ ID NO: 4) after amino acid 142 and a new stop codon (TGA) that is 2 bp downstream from the original stop codon (TAA) {see Figures 2(b) and (d)). The mutation at amino acid 142 leads to loss of all conserved motifs, indicating that dlt is a knockout mutant {see Figure 2(d)). RT-PCR analysis showed that aberrant DLT transcripts are expressed in the dlt plant {see Figure 2(c)).
[00131] Example 3
[00132] Complementation Test
[00133] To confirm whether the DLT mutation was responsible for the dlt phenotype, a genomic fragment of 7577 bp containing the entire DLT coding sequence, 3253 bp of the 5' upstream region and 2471 bp of the 3' downstream region was digested from the BAC clone OSJNBa0038F22( Arizona Genomics Institute) using restriction enzymes BamHI and Kpnl (Promega). The fragment was recovered, ligated with a binary vector pCAMBIA1300 (Cambia, Australia), and transformed into Agrobacterium AGLl (ATCC). The transformed AGLl was used to infect the callus of a dit mutant as described, for example, in Yi et al., Journal of Genetics and Genomics, 28(4):352-358 (2001).
[00134] After obtaining the regenerated plants by hygromycin selection, PCR amplification was performed on the genomic DNA of the regenerated plants using forward (5'- CATCAATCCATTGCAGGGACGAT-3' (SEQ ID NO: 5)) and reverse (5'- CGTTGAGCGTGAAGTGCAGGAA-S' (SEQ ID NO: 6) primers flanking the 62 bp deleted segment of the DLT gene. Forty positive transgenic plants were identified {see Figure 3(c)). The phenotype of these transgenic plants in both the vegetative and productive phase totally reverted to that of the wild type {see Figures 3(a) and (b)), demonstrating that the 62 bp deletion in DLT was responsible for the dlt mutant phenotype.
[00135] Example 4
[00136] Comparison of mutant and wild type sensitivity to 24-epibrassinolide
[00137] Seeds of the wild type rice Zhonghua 11 (Institute of Genetics and Developmental Biology, CAS) and the dlt mutant were germinated for two days at 30 °C, and seeds having the same germination stage/condition were sown into water. After incubating for 3 days at 30 °C and exposed to 10 hours of light per day, plants were spotted on top of the second lamina with lμl ethanol in which either 0 ng, 10 ng, 100 ng or 1000 ng of 24-epi-brassinolide (E- 1641; Sigma) was dissolved.
[00138] After another 3 days of growth under the same conditions, the angles between the lamina and the leaf sheath were compared. As shown in Figure 4(a), the dlt mutant had no substantial response to different concentrations of 24-epi-brassinolide. In contrast, the wild type plants were comparatively more sensitive, as the lamina joint angle of wild type plants increased incrementally with higher concentrations of 24-epi-brassinolide.
[00139] Using a different method (see e.g., Wada et al, Plant Cell Physiol., 22:323-325 (1981)), wild type and dlt mutant seeds were germinated at 30°C for two days, and seeds having the same germination stage/condition were sown onto a screen immersed in water and grown for 8 days in darkness at 30 °C. Segments from the top of the plant, comprising one leaf blade (1 cm) and one leaf sheath (1 cm), were cut and floated on distilled water for 24 hours. [00140] Segments were subsequently incubated in 2.5 mmol/L potassium maleate solution without or with 5 ng/ml (final concentration) 24-epi-brassinolide for 48 hours, after which the lamina joint angles were compared. As shown in Figure 4(b), the dlt mutant is clearly less sensitive to 24-epi-brassinolide than wild type plants.
[00141] A third brassinosteroid response assay was performed to assess the effect of 24-epi- brassinolide on coleoptile elongation in dlt mutants (see e.g., Yamamuro et al., 2000). Seeds were germinated and grown on 0.7% agar medium supplemented with various concentrations of 24-epiBL. Comparison of coleoptile length showed that the dlt mutant has a much lower response to BL than WT does (see Figure 4(c)).
[00142] Example 5
[00143] DLT expression
[00144] RNAs were extracted from various organs and tissues of the dlt mutant, using MMLV reverse transcriptase (Promega) to perform reverse transcription. Real-time fluorescence quantitative PCR was subsequently performed to detect the expression of DLT. Rice ACTLNl was used as the internal control, and SYBR Green I was used as the dye. Primers 5'- TGCGGATACTCAACGCCATC A-3' (forward; SEQ ID NO: 7) and 5'- ACTCGCCGACTCCGGTGATC-3' (reverse; SEQ ID NO: 8) were used to amplify DLT, and primers 5'-AGCAACTGGGATGATATGGA-S' (forward; SEQ ID NO: 9) and 5'-
CAGGGCGATGTAGGAAAGC-3' (reverse; SEQ ID NO: 10) were used to amplify ACTINl.
The experiment was performed in triplicate.
[00145] As demonstrated in Figure 5(a), the expression level of DLT in panicle, stem and root were relatively high, while the level in leaf was low. Among internodes, the expression level of
DLT 'in the second internode was lower than the others {see Figure 5(b)). This expression pattern was consistent with the specifically shortened length of the second internode and also consistent with the expression pattern of OsBRI reported by Yamamuro et al {Plant Cell, 12:1591-1606
(2000)).
[00146] To better understand the DLT expression pattern, approximately 2 kb of the DLT 5' region was amplified and introduced into the pCAMBIA1391Z vector, resulting in the PRODLT
:GUS construct. Analysis of transgenic plants harboring the PRODLT:GUS construct confirmed the universal expression of DLT in various tissues but preferentially in actively dividing and elongating cells {see Figure 6).
[00147] GUS staining showed that, in young seedlings, DLT is mainly expressed in the shoot apical meristem and elongating cells (Figure 6, panes 1 and 2). In leaves, DLT is expressed at significantly higher levels in unexpanded leaves than in green functional leaves, although with a slightly preferential expression in the leaf joint (Figure 6, panes 3-5). DLT expression could not be detected in mature leaves by GUS staining.
[00148] In culm tissues, DLT is expressed strongly in the internodes prior to rapid elongation
(Figure 6, pane 6). However, during the elongation period of the internode, expression of DLT is confined to the lower part, presumably corresponding to the division and elongating zones
(Figure 6, pane 7). Moreover, at the end of the elongation process, GUS activity was only detected in the very low part of the internode, namely the division zone (Figure 6, pane 8).
Cross-sections showed that DLT expression in the stem is preferable in ground tissues (Figure 6, panes 9 and 10). DLT was also found to be expressed very strongly in young panicles and gradually became weak in the floret (Figure 6, panes 11 and 12). In the root, the DZTpromoter was highly active in both primary root and lateral root, with much stronger activity in root tips
(Figure 6, panes 2 and 14).
[00149] DLT also showed higher expression in the vascular cylinder and lateral root outgrowth locations, but much lower expression in cortex tissues (Figure 6, panes 13 and 14). These expression patterns correlate well with DLTs putative function in cell elongation and division. Young tissues are those in which cells are actively dividing and elongating, and this is also where BR functions are most active.
[00150] Example 6
[00151] Negative regulation of DLT expression by 24-epi-brassinolide
[00152] One week-old wild type seedlings were sprayed with 1 μmol/L 24-epi-brassinolide. RNA was extracted at different time points after spraying and reverse transcribed to detect the expression of DLT using real-time quantitative PCR methodology described in Example 4. The experiment was performed in triplicate. As shown in Figure 7(a), DL T expression decreased gradually after exposure to 24-epi-brassinolide and was reduced to about 40% of pre-exposure levels 12 hours later.
[00153] The expression pattern of DLT in the brassinosteroid-synthesizing mutants d.2-1 and dll-2 was also evaluated using the same procedure described above. d.2-1 is a dwarf mutant exhibiting a pleiotropic abnormal phenotype similar to that of the rice brassinosteroid-insensitive mutant, d61. Hong et al. concluded that the D2 gene encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway {see Hong et al., Plant Cell, 15:2900-2910 (2003)). Similarly, dll-2 is a dwarf mutant that bears small round grains. Tanabe et al. concluded that the DIl gene also encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway {see Tanabe et al., Plant Cell, 17:776-790 (2005)). [00154] The experiment was performed in triplicate. As shown in Figure 7(b), DLT expression in d.2-1 and dll-2 plants was significantly greater than in wild type Shiokari plants. The observations from these two brassinosteroid-synthesizing mutants further reinforce the conclusion that brassinosteroids negatively regulate the expression of DLT
[00155] Example 7
[00156] Expression of brassinosteroid-related genes in the dlt mutant
[00157] Fluorescence quantitative PCR was used to determine the expression levels of several known brassinosteroid synthesizing genes {D2, DIl, OsCPD and OsBRόox) and two genes
{OsXTRl and OsBLEI) which are located downstream of the brassinosteroid signaling and are induced by brassinosteroids in the dlt mutant. Sequences of the primers used to amplify D2, DIl and OsCPD can be found in Shimada et al {Plant J., 48:390-402 (2006)). To amplify OsBRόox, a forward primer having the sequence 5'-CAGGTACGGGAGCGTGTT -3' (SEQ ID NO: 11) and a reverse primer having the sequence 5'-TGAAGCCTTGGTAGTAGTTGGT-S' (SEQ ID NO: 12) were used. Primer sequences used to amplify OsXTRl are disclosed in Duan et al (Plant J., 47:519-531 (2006)) and primer sequences used to amplify OsBLE2 are disclosed in Yang et al (Plant MoI. Biol., 52:843-854 (2003)). The expression level of each gene in the wild type Zhonghua 11 was set as 1, and the ratio of the expression level in the dlt mutant to that in the wild type was calculated. The experiment was carried out in triplicate. [00158] As shown in Figure 8, transcripts of D2, DIl, OsCPD and OsBRόox significantly accumulated in the dlt mutant, whereas OsXTRl and OsBLE2 expression was downregulated. These observations support the premise that DLT plays a role in brassinosteroid signaling in rice.
[00159] Example 8
[00160] Interaction of DL T promoter with OsBZRl
[00161] As brassinosteroids (BRs) repress DLT expression and as three brassinosteroid response elements (BRREs) were identified within the 600 bp genomic sequence upstream of the DLT transcript (see Figure 8(a)), an electrophoretic mobility shift assay was performed to test whether OsBZRl binds to these elements. The OsBZRl coding region was cloned into a maltose binding protein (MBP) fusion vector (pET-MALc-H vector) using primers Os-BES lNAsp718 (5'-CTCGGTACCGGAGCTGGTGGGTATGACGTC-S'; SEQ ID NO: 13) and OsBESlCHind3 (5'-CGCAAGCTTTCATTTCGCGCCGACGCCGAGC-S'; SEQ ID NO: 14). The recombinant MBP-OsBZRl was purified from E. coli using amylose resin (NEB). Wild type oligos derived from the DLT promoter, and mutant forms in which each nucleotide in a brassinosteroid response element (CGTGCG; SEQ ID NO: 15) was replaced with an adenosine were synthesized and annealed. The WT probe was labeled with 32P-C-ATP, and approximately 0.5 ng of probe was used for each binding assay. For competition experiments, excess unlabeled probe was added to the reactions at indicated molar ratios compared to labeled probe.
[00162] As shown in Figure 9, OsBZRl can bind to the labeled WT probe and unlabeled WT probe competes for binding with OsBRZl. OsBZRl does not bind to the mutant probe in which the BRRE is altered as described above. These results demonstrate that OsBZRl can bind to the DLT promoter through the BRRE. [00163] Example 9
[00164] Increased tiller number in rice transformed with DLT
[00165] The open reading frame OfDLT(SEQ ID NO: l; nt 771-2621 of SEQ ID NO: 18; ), was amplified from rice genomic DNA using 5'-
CCATGGATGTTGGCGGGTTGCTCGTTCTCGT-3' (SEQ ID NO: 16) as the forward primer and 5'-AGATCTGATGTTGGCGGGTTGCTCGTTCTCGT-S' (SEQ ID NO: 17) as the reverse primer. Restriction sites for Noel and BgIII were added to the end of the forward and reverse primer, respectively. The PCR product was recovered and ligated into the pMD18-T vector (Takara Bio Inc.). After confirmation by sequencing, the vector was digested by Ncol and BgIII. The resultant fragment was ligated into a binary expression vector pCAMBIA1302 (Cambia), resulting in a recombinant expression vector pCAMBIAl 302-DLT.
[00166] The recombinant expression vector was transformed into the callus of the rice cultivar Zhonghua 11 via an Agrobacterium AGl (ATCC) mediated method. Ten transgenic T0 generation plants were obtained by resistance screening.
[00167] RNAs were extracted from the ten To generation transgenic positive plants and expression levels of DLT were determined by quantitative fluorescence PCR as described previously. The DLT expression level in wild type rice Zhonghua 11 was set as 1 and the ratio of the DLT expression level in each of the transgenic plants to that of the wild type was calculated. The experiments were performed in triplicate.
[00168] As shown in Figure 10(a), seven of the T0 generation transgenic plants had significantly upregulated DLT gene expression. Of these seven plants, five of them had more than 100-fold increase in DL T expression levels as compared to their Zhonghua 11 counterparts. These five plants exhibited a curly leaf blade, enlarged lamina joint angle, increased tiller number and slight dwarfism (see plant no. 5 in Figure 10(b)). The other two T0 transgenic plants, which had less than 100-fold increases in expression of DLT, exhibited significantly increased tiller numbers and slightly increased height, (see plant no. 10 in Figure 10(b)). These observations indicate that increased DLT expression leads to increased tiller number in rice. [00169] Additional statistical analysis was performed on ten T2 plants during the heading stage. As shown in Figure 11, most of the transgenic plants had increased tiller number compared to the corresponding wild type plants. Moreover, for those plants in which DLT overexpression caused no obvious change in leaf morphology (i.e., plant lines 1, 2, 4, 9 and 10, which correspond to the like numbered plant lines shown in Figure 10(a)), the increase in tiller number was proportional to the increase in DLT expression. However, very high expression levels of DLT resulted in changes in morphology in certain plants (i.e., plant lines 3, 5, 6, 7 and 8, which correspond to the like numbered plant lines shown in Figure 10(a)). The leaves in these plants narrowed and rolled inward, and the lamina joint bending angle increased. The extent of these changes were proportional to DL T expression levels, and a decrease in tiller number (as compared to plant line 1) was also observed. Without wishing to be bound by theory, the reduction in tiller number is believed to be caused indirectly by a functional defect in leaf morphology.
[00170] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[00171] While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.
63367.0030.1 SEQL_ST25 SEQUENCE LISTING
<110> insti tute of Geneti cs and Developmental Bi ol ogy , Chi nese Academy of Sci ences Syngenta Parti ci pations AG Chu , Chengcai Tong , Hongni ng Ji n , Yun Li u , Wen bo Li , Feng Fang , Jun Zhu , Li huang
<120> Genes Associ ated wi th Pl ant Ti l l er Number and Uses Thereof <130> 63367.0030.1
<140> Not yet assi gned <141> 2009-12-29
<150> CN 200810247366. 3 <151> 2008-12-30
<160> 18
<170> Patentln version 3.5
<210> 1
<211> 1851
<212> DNA
<213> Oryza sativa
<400> 1 atgttggcgg gttgctcgtt ctcgtcgtcg aggcatcaga tgagcaccgc gcagcgtttc 60 gacatcctcc cctgcggctt ctccaagcgc ggcagccgcg gcgacggcgc cgccccgcgg 120 gtcgccggcg acgccaggag cggcgccacc acctgctcct tccggacgca ccccgcgccg 180 ccggtcaccc agtccgtgtc ctggggcgcc aagccggagc ccggcggcaa tggcaatggc 240 gcccaccgcg ccgttaagcg ggcgcatgac gaggacgcgg tcgaggagta tggccccatt 300 gttcgcgcca agcggacgcg gatgggcggc gacggcgatg aggtatggtt ccatcaatcc 360 attgcaggga cgatgcaagc gacggcggcg ggagaaggag aggaggcgga ggaggagaag 420 gtcttcttgg tgccgagcgc ggcggcgttc ccgcacggca tggccgccgc ggggccatcg 480 ctggccgcgg ccaagaagga ggagtacagc aagtcgccgt ccgactcgtc gtcctcgtcg 540 ggcacggacg gcggctcgtc ggcgatgatg ccgccgccgc agccgcccga gttcgacgcg 600 aggaacggcg tgccggcgcc ggggcaggcg gagcgggagg cgctggagct ggtgcgcgcg 660 ctcaccgcgt gcgccgactc cctctccgcc ggcaaccacg aggccgccaa ctactacctg 720 gcccggctcg gcgagatggc ctcgccggcg gggcccacgc cgatgcaccg cgtggccgcc 780 63367.0030.1 SEQL_ST25 tacttcaccg aggcgctcgc gctccgcgtc gtgcgcatgt ggccgcacat gttcgacatc 840 ggcccgccgc gggagctcac cgacgacgcc ttcggcggcg gcgacgacga cgccatggcg 900 ctgcggatac tcaacgccat cacgcccatc ccgaggttcc tgcacttcac gctcaacgag 960 cgcctcctcc gcgagttcga ggggcacgag cgcgtccacg tcatcgactt cgacatcaag 1020 caggggctcc aatggccggg cttgctccag agcctggccg cgcgggcggt gcctccggcg 1080 cacgtgcgga tcaccggagt cggcgagtcg aggcaggagc tgcaggagac gggagcgcgg 1140 ctggcgcgcg tcgccgccgc gctcggcctg gcgttcgagt tccacgccgt ggtcgaccgg 1200 ctcgaggacg tccgcctgtg gatgctccac gtcaagcgcg gcgagtgcgt ggccgtgaac 1260 tgcgtcctcg ccatgcaccg cctgctccgc gacgacgccg cgctgaccga cttcctgggg 1320 ctagcgcgca gcacgggcgc caccatcctc ctcctcggcg agcacgaggg cggcggcctc 1380 aactcgggga ggtgggaggc gcggttcgcg cgcgcgctgc ggtactacgc cgcggcgttc 1440 gacgcggtgg acgcggcggg gctgccggag gcgagccccg cgagggccaa ggcggaggag 1500 atgttcgcgc gggagatccg caacgcggtg gcgttcgagg gccccgagcg gttcgagcgc 1560 cacgagagct tcgccgggtg gcggcggcgc atggaggacg gcggcgggtt caagaacgcc 1620 ggcatcggcg agcgcgaggc gatgcagggg cgcatgatcg cgaggatgtt cgggccggac 1680 aagtacaccg tgcaggcgca cggcggcggc ggcagcggcg gcggcgaggc gctcacgctc 1740 cggtggctgg accagccgct gtacaccgtg acggcgtgga cgccggcggg cgacggcgcg 1800 ggaggcagca ccgtgtcggc gtccacaaca gcatcacatt ctcagcaaag c 1851
<210> 2
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<213> Oryza sativa
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Met Leu Al a Gl y Cys Ser Phe Ser ser ser Arg Hi s Gi n Met Ser Thr 1 5 10 15
Al a Gi n Arg Phe Asp li e Leu Pro Cys Gly Phe Ser Lys Arg Gl y Ser 20 25 30
Arg Gly Asp Gl y Al a Al a Pro Arg VaI Al a Gly Asp Al a Arg ser Gly 35 40 45
Al a Thr Thr Cys Ser Phe Arg Thr Hi s Pro Al a Pro Pro VaI Thr Gi n 63367.0030.1 SEQL_ST25 50 55 60
Ser VaI Ser Trp Gly Ala Lys Pro Gl u Pro Gly Gly Asn Gly Asn Gly 65 70 75 80
Ala His Arg Ala VaI Lys Arg Ala His Asp Gl u Asp Ala VaI Gl u Gl u 85 90 95
Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110
Asp Glu VaI Trp Phe His Gin ser lie Ala Gly Thr Met Gin Ala Thr 115 120 125
Ala Ala Gly Glu Gly Glu Glu Ala Glu Glu Glu Lys VaI Phe Leu VaI 130 135 140
Pro Ser Ala Ala Ala Phe Pro His Gly Met Ala Ala Ala Gly Pro Ser 145 150 155 160
Leu Ala Ala Ala Lys Lys Glu Glu Tyr ser Lys Ser Pro Ser Asp Ser 165 170 175
Ser Ser ser ser Gly Thr Asp Gly Gly Ser ser Ala Met Met Pro Pro 180 185 190
Pro Gin Pro Pro Glu Phe Asp Ala Arg Asn Gly VaI Pro Ala Pro Gly 195 200 205
Gin Ala Glu Arg Glu Ala Leu Glu Leu VaI Arg Ala Leu Thr Ala Cys 210 215 220
Ala Asp Ser Leu Ser Ala Gly Asn His Glu Ala Ala Asn Tyr Tyr Leu 225 230 235 240
Ala Arg Leu Gly Glu Met Ala Ser Pro Ala Gly Pro Thr Pro Met His 245 250 255
Arg VaI Ala Ala Tyr Phe Thr Glu Ala Leu Ala Leu Arg VaI VaI Arg 260 265 270
Met Trp Pro His Met Phe Asp lie Gly Pro Pro Arg Glu Leu Thr Asp 275 280 285 63367.0030.1 SEQL_ST25
Asp Ala Phe Gly Gly Gly Asp Asp Asp Ala Met Ala Leu Arg lie Leu 290 295 300
Asn Ala lie Thr Pro lie Pro Arg Phe Leu His Phe Thr Leu Asn Glu 305 310 315 320
Arg Leu Leu Arg Glu Phe Glu Gly His Glu Arg VaI His VaI lie Asp 325 330 335
Phe Asp lie Lys Gin Gly Leu Gin Trp Pro Gly Leu Leu Gin ser Leu 340 345 350
Ala Ala Arg Ala VaI Pro Pro Ala His VaI Arg lie Thr Gly VaI Gly 355 360 365
Glu Ser Arg Gin Glu Leu Gin Glu Thr Gly Ala Arg Leu Ala Arg VaI 370 375 380
Ala Ala Ala Leu Gly Leu Ala Phe Glu Phe His Ala VaI VaI Asp Arg 385 390 395 400
Leu Glu Asp VaI Arg Leu Trp Met Leu His VaI Lys Arg Gly Glu Cys 405 410 415
VaI Ala VaI Asn cys VaI Leu Ala Met His Arg Leu Leu Arg Asp Asp 420 425 430
Ala Ala Leu Thr Asp Phe Leu Gly Leu Ala Arg ser Thr Gly Ala Thr 435 440 445
lie Leu Leu Leu Gly Glu His Glu Gly Gly Gly Leu Asn ser Gly Arg 450 455 460
Trp Glu Ala Arg Phe Ala Arg Ala Leu Arg Tyr Tyr Ala Ala Ala Phe 465 470 475 480
Asp Ala VaI Asp Ala Ala Gly Leu Pro Glu Ala Ser Pro Ala Arg Ala 485 490 495
Lys Ala Glu Glu Met Phe Ala Arg Glu lie Arg Asn Ala VaI Ala Phe 500 505 510
Glu Gly Pro Glu Arg Phe Glu Arg His Glu Ser Phe Ala Gly Trp Arg 63367.0030.1 SEQL_ST25 515 520 525
Arg Arg Met Gl u Asp Gl y Gl y Gl y Phe Lys Asn Al a Gl y li e Gl y Gl u 530 535 540
Arg Gl u Al a Met Gi n Gly Arg Met li e Al a Arg Met Phe Gl y Pro Asp 545 550 555 560
Lys Tyr Thr VaI Gi n Al a Hi s Gly Gl y Gly Gly Ser Gly Gl y Gl y Gl u 565 570 575
Al a Leu Thr Leu Arg Trp Leu Asp Gi n Pro Leu Tyr Thr VaI Thr Al a 580 585 590
Trp Thr Pro Al a Gly Asp Gl y Al a Gl y Gly Ser Thr VaI Ser Al a Ser 595 600 605
Thr Thr Al a Ser Hi s Ser Gi n Gi n Ser 610 615
<210> 3
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<213> Oryza sati va
<400> 3 atgttggcgg gttgctcgtt ctcgtcgtcg aggcatcaga tgagcaccgc gcagcgtttc 60 gacatcctcc cctgcggctt ctccaagcgc ggcagccgcg gcgacggcgc cgccccgcgg 120 gtcgccggcg acgccaggag cggcgccacc acctgctcct tccggacgca ccccgcgccg 180 ccggtcaccc agtccgtgtc ctggggcgcc aagccggagc ccggcggcaa tggcaatggc 240 gcccaccgcg ccgttaagcg ggcgcatgac gaggacgcgg tcgaggagta tggccccatt 300 gttcgcgcca agcggacgcg gatgggcggc gacggcgatg aggtatggtt ccatcaatcc 360 attgcaggga cgatgcaagc gacggcggcg ggagaaggag aggaggcgga ggaggagaag 420 gtcttcttgg tgccgagcgc ggcggcgttc ccgcacggca tggccgccgc ggggccatcg 480 ctggccgcgg ccaagaagga ggagtacagc aagtcgccgt ccgcccgagt tcgacgcgag 540 gaacggcgtg ccggcgccgg ggcaggcgga gcgggaggcg ctggagctgg tgcgcgcgct 600 caccgcgtgc gccgactccc tctccgccgg caaccacgag gccgccaact actacctggc 660 ccggctcggc gagatggcct cgccggcggg gcccacgccg atgcaccgcg tggccgccta 720 cttcaccgag gcgctcgcgc tccgcgtcgt gcgcatgtgg ccgcacatgt tcgacatcgg 780 63367.0030.1 SEQL_ST25 cccgccgcgg gagctcaccg acgacgcctt cggcggcggc gacgacgacg ccatggcgct 840 gcggatactc aacgccatca cgcccatccc gaggttcctg cacttcacgc tcaacgagcg 900 cctcctccgc gagttcgagg ggcacgagcg cgtccacgtc atcgacttcg acatcaagca 960 ggggctccaa tggccgggct tgctccagag cctggccgcg cgggcggtgc ctccggcgca 1020 cgtgcggatc accggagtcg gcgagtcgag gcaggagctg caggagacgg gagcgcggct 1080 ggcgcgcgtc gccgccgcgc tcggcctggc gttcgagttc cacgccgtgg tcgaccggct 1140 cgaggacgtc cgcctgtgga tgctccacgt caagcgcggc gagtgcgtgg ccgtgaactg 1200 cgtcctcgcc atgcaccgcc tgctccgcga cgacgccgcg ctgaccgact tcctggggct 1260 agcgcgcagc acgggcgcca ccatcctcct cctcggcgag cacgagggcg gcggcctcaa 1320 ctcggggagg tgggaggcgc ggttcgcgcg cgcgctgcgg tactacgccg cggcgttcga 1380 cgcggtggac gcggcggggc tgccggaggc gagccccgcg agggccaagg cggaggagat 1440 gttcgcgcgg gagatccgca acgcggtggc gttcgagggc cccgagcggt tcgagcgcca 1500 cgagagcttc gccgggtggc ggcggcgcat ggaggacggc ggcgggttca agaacgccgg 1560 catcggcgag cgcgaggcga tgcaggggcg catgatcgcg aggatgttcg ggccggacaa 1620 gtacaccgtg caggcgcacg gcggcggcgg cagcggcggc ggcgaggcgc tcacgctccg 1680 gtggctggac cagccgctgt acaccgtgac ggcgtggacg ccggcgggcg acggcgcggg 1740 aggcagcacc gtgtcggcgt ccacaacagc atcacattct cagcaaagct aagc 1794
<210> 4
<211> 598
<212> PRT
<213> Oryza sativa
<400> 4
Met Leu Al a Gl y Cys Ser Phe Ser ser ser Arg Hi s Gi n Met Ser Thr 1 5 10 15
Al a Gi n Arg Phe Asp li e Leu Pro Cys Gly Phe Ser Lys Arg Gl y Ser 20 25 30
Arg Gly Asp Gl y Al a Al a Pro Arg VaI Al a Gly Asp Al a Arg ser Gly 35 40 45
Al a Thr Thr Cys Ser Phe Arg Thr Hi s Pro Al a Pro Pro VaI Thr Gi n 50 55 60 63367.0030.1 SEQL_ST25
Ser VaI Ser Trp Gly Ala Lys Pro Gl u Pro Gly Gly Asn Gly Asn Gly 65 70 75 80
Ala His Arg Ala VaI Lys Arg Ala His Asp Gl u Asp Ala VaI Gl u Gl u 85 90 95
Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110
Asp Glu VaI Trp Phe His Gin ser lie Ala Gly Thr Met Gin Ala Thr 115 120 125
Ala Ala Gly Glu Gly Glu Glu Ala Glu Glu Glu Lys VaI Phe Leu VaI 130 135 140
Pro Ser Ala Ala Ala Phe Pro His Gly Met Ala Ala Ala Gly Pro Ser 145 150 155 160
Leu Ala Ala Ala Lys Lys Glu Glu Tyr ser Lys Ser Pro Ser Ala Arg 165 170 175
VaI Arg Arg Glu Glu Arg Arg Ala Gly Ala Gly Ala Gly Gly Ala Gly 180 185 190
Gly Ala Gly Ala Gly Ala Arg Ala His Arg VaI Arg Arg Leu Pro Leu 195 200 205
Arg Arg Gin Pro Arg Gly Arg Gin Leu Leu Pro Gly Pro Ala Arg Arg 210 215 220
Asp Gly Leu Ala Gly Gly Ala His Ala Asp Ala Pro Arg Gly Arg Leu 225 230 235 240
Leu His Arg Gly Ala Arg Ala Pro Arg Arg Ala His VaI Ala Ala His 245 250 255
VaI Arg His Arg Pro Ala Ala Gly Ala His Arg Arg Arg Leu Arg Arg 260 265 270
Arg Arg Arg Arg Arg His Gly Ala Ala Asp Thr Gin Arg His His Ala 275 280 285
His Pro Glu VaI Pro Ala Leu His Ala Gin Arg Ala Pro Pro Pro Arg 63367.0030.1 SEQL_ST25 290 295 300
VaI Arg Gly Ala Arg Ala Arg Pro Arg His Arg Leu Arg His Gin Ala 305 310 315 320
Gly Ala Pro Met Ala Gly Leu Ala Pro Glu Pro Gly Arg Ala Gly Gly 325 330 335
Ala Ser Gly Ala Arg Ala Asp His Arg ser Arg Arg VaI Glu Ala Gly 340 345 350
Ala Ala Gly Asp Gly Ser Ala Ala Gly Ala Arg Arg Arg Arg Ala Arg 355 360 365
Pro Gly VaI Arg VaI Pro Arg Arg Gly Arg Pro Ala Arg Gly Arg Pro 370 375 380
Pro VaI Asp Ala Pro Arg Gin Ala Arg Arg VaI Arg Gly Arg Glu Leu 385 390 395 400
Arg Pro Arg His Ala Pro Pro Ala Pro Arg Arg Arg Arg Ala Asp Arg 405 410 415
Leu Pro Gly Ala Ser Ala Gin His Gly Arg His His Pro Pro Pro Arg 420 425 430
Arg Ala Arg Gly Arg Arg Pro Gin Leu Gly Glu VaI Gly Gly Ala VaI 435 440 445
Arg Ala Arg Ala Ala VaI Leu Arg Arg Gly VaI Arg Arg Gly Gly Arg 450 455 460
Gly Gly Ala Ala Gly Gly Glu Pro Arg Glu Gly Gin Gly Gly Gly Asp 465 470 475 480
VaI Arg Ala Gly Asp Pro Gin Arg Gly Gly VaI Arg Gly Pro Arg Ala 485 490 495
VaI Arg Ala Pro Arg Glu Leu Arg Arg VaI Ala Ala Ala His Gly Gly 500 505 510
Arg Arg Arg VaI Gin Glu Arg Arg His Arg Arg Ala Arg Gly Asp Ala 515 520 525 63367.0030.1 SEQL_ST25
Gly Ala His Asp Arg Glu Asp VaI Arg Ala Gly Gin VaI His Arg Ala 530 535 540
Gly Ala Arg Arg Arg Arg Gin Arg Arg Arg Arg Gly Ala His Ala Pro 545 550 555 560
VaI Ala Gly Pro Ala Ala VaI His Arg Asp Gly VaI Asp Ala Gly Gly 565 570 575
Arg Arg Arg Gly Arg Gin His Arg VaI Gly VaI His Asn Ser lie Thr 580 585 590
Phe Ser Ala Lys Leu Ser 595
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 5 catcaatcca ttgcagggac gat 23
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 6 cgttgagcgt gaagtgcagg aa 22
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 7 tgcggatact caacgccatc a 21
<210> 8 63367.0030.1 SEQL_ST25
<211> 20
<212> DNA
<213> Artifi cial Sequence
<220>
<223> Primer
<400> 8 actcgccgac tccggtgatc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 9 agcaactggg atgatatgga 20
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 10 cagggcgatg taggaaagc 19
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 11 caggtacggg agcgtgtt 18
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 12 tgaagccttg gtagtagttg gt 22 63367.0030.1 SEQL_ST25
<210> 13
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 13 cgcaagcttt catttcgcgc cgacgccgag c 31
<210> 14
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 14 cgcaagcttt catttcgcgc cgacgccgag c 31
<210> 15
<211> 6
<212> DNA
<213> Artificial Sequence
<220>
<223> Brassinosteroid response element
<400> 15 cgtgcg 6
<210> 16
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 16 ccatggatgt tggcgggttg ctcgttctcg t 31
<210> 17
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 17 63367.0030.1 SEQL_ST25 agatctgatg ttggcgggtt gctcgttctc gt 32
<210> 18
<211> 3084
<212> DNA
<213> Oryza sativa
<400> 18 gagtgagagt gagagagtga gagcagagac caccaccacc ggagaggtta gtgagagagg 60 agtggtaatg gtgaggcaac aagagtaggt tccatttcat atcatcacta ggatagcgta 120 gtttgtaggc tgcatctcca tctccatcgc cattgattcg cattgcatcc atcattttag 180 gatgttctac tagggttctt gatttttctt ttggtttgtt gttttgacga atggaggtat 240 tgttgggatt cgccgcctgc tgctcgtcgt cgtcgtcgcc gatgaggagg ccgtgcgggc 300 tctgccccgg catgtccgat cgttcgtgat ttgttttttc tacatgtttt agggcccatt 360 tgttcttgat cctattcttt gattcttttg tactaagcat tctaaggcga agccacccat 420 tctttcctgc atatatactt acaaacacat agcccccatc tgatctcaca aacattattt 480 ctctctcttt ttttctcagt tttttctttg ttgatttact gaccaaattc tttggaagaa 540 caacaagatc atctggtttt tatctgctca ttcttttgta catcgaatca tatacatttc 600 cattccacca aagccttagc cagataccac agagagagtg tgagagaaat cagagtgaga 660 aacagaggag gaagaagaag aagaagacga ggaggaggag gaggaggagc aggaggagga 720 ggaggtctct tcttggcacg tcgcgttccg gcgagtgacg tgtctccggg atgttggcgg 780 gttgctcgtt ctcgtcgtcg aggcatcaga tgagcaccgc gcagcgtttc gacatcctcc 840 cctgcggctt ctccaagcgc ggcagccgcg gcgacggcgc cgccccgcgg gtcgccggcg 900 acgccaggag cggcgccacc acctgctcct tccggacgca ccccgcgccg ccggtcaccc 960 agtccgtgtc ctggggcgcc aagccggagc ccggcggcaa tggcaatggc gcccaccgcg 1020 ccgttaagcg ggcgcatgac gaggacgcgg tcgaggagta tggccccatt gttcgcgcca 1080 agcggacgcg gatgggcggc gacggcgatg aggtatggtt ccatcaatcc attgcaggga 1140 cgatgcaagc gacggcggcg ggagaaggag aggaggcgga ggaggagaag gtcttcttgg 1200 tgccgagcgc ggcggcgttc ccgcacggca tggccgccgc ggggccatcg ctggccgcgg 1260 ccaagaagga ggagtacagc aagtcgccgt ccgactcgtc gtcctcgtcg ggcacggacg 1320 gcggctcgtc ggcgatgatg ccgccgccgc agccgcccga gttcgacgcg aggaacggcg 1380 tgccggcgcc ggggcaggcg gagcgggagg cgctggagct ggtgcgcgcg ctcaccgcgt 1440 gcgccgactc cctctccgcc ggcaaccacg aggccgccaa ctactacctg gcccggctcg 1500 63367.0030.1 SEQL_ST25 gcgagatggc ctcgccggcg gggcccacgc cgatgcaccg cgtggccgcc tacttcaccg 1560 aggcgctcgc gctccgcgtc gtgcgcatgt ggccgcacat gttcgacatc ggcccgccgc 1620 gggagctcac cgacgacgcc ttcggcggcg gcgacgacga cgccatggcg ctgcggatac 1680 tcaacgccat cacgcccatc ccgaggttcc tgcacttcac gctcaacgag cgcctcctcc 1740 gcgagttcga ggggcacgag cgcgtccacg tcatcgactt cgacatcaag caggggctcc 1800 aatggccggg cttgctccag agcctggccg cgcgggcggt gcctccggcg cacgtgcgga 1860 tcaccggagt cggcgagtcg aggcaggagc tgcaggagac gggagcgcgg ctggcgcgcg 1920 tcgccgccgc gctcggcctg gcgttcgagt tccacgccgt ggtcgaccgg ctcgaggacg 1980 tccgcctgtg gatgctccac gtcaagcgcg gcgagtgcgt ggccgtgaac tgcgtcctcg 2040 ccatgcaccg cctgctccgc gacgacgccg cgctgaccga cttcctgggg ctagcgcgca 2100 gcacgggcgc caccatcctc ctcctcggcg agcacgaggg cggcggcctc aactcgggga 2160 ggtgggaggc gcggttcgcg cgcgcgctgc ggtactacgc cgcggcgttc gacgcggtgg 2220 acgcggcggg gctgccggag gcgagccccg cgagggccaa ggcggaggag atgttcgcgc 2280 gggagatccg caacgcggtg gcgttcgagg gccccgagcg gttcgagcgc cacgagagct 2340 tcgccgggtg gcggcggcgc atggaggacg gcggcgggtt caagaacgcc ggcatcggcg 2400 agcgcgaggc gatgcagggg cgcatgatcg cgaggatgtt cgggccggac aagtacaccg 2460 tgcaggcgca cggcggcggc ggcagcggcg gcggcgaggc gctcacgctc cggtggctgg 2520 accagccgct gtacaccgtg acggcgtgga cgccggcggg cgacggcgcg ggaggcagca 2580 ccgtgtcggc gtccacaaca gcatcacatt ctcagcaaag ctaagctgac gatgaatggt 2640 gattaggtga agagaaagaa agaacaaagc ctttttttac agtgcttctt ttgttaatga 2700 tgattagttc atacagtatg acaattcttt tatacattca gagaaaagaa agaagaaaga 2760 aaggtgtagt tttttgtttt atagattgat aggtggaaag attcaattaa atcaaattca 2820 attcaatttt tagattgtaa ttctttataa atattctttt ggctgttgag agagagtccc 2880 ctgcaaaatg tagctgcatg tagaagaaag aaagcaaaga agcagtagat agattagcag 2940 gggcagcatc tctcacagtc actattagtg tctccggctg ttattataca acattattat 3000 tacaatcaaa ttctttcatc attcattcta catgtaatct ctgttcagaa tcagaatgaa 3060 atgaaacatg tgttatattt ctcc 3084

Claims

Attorney Ref.: 63367.0030.1 CLAIMS What is claimed is:
1. An isolated DLT polynucleotide selected from the group consisting of:
(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3;
(b) a nucleic acid comprising a nucleotide sequence at least 70% identical to (a);
(c) a nucleic acid that specifically hybridizes to the complement of (a) under stringent hybridization conditions;
(d) a nucleic acid comprising an open reading frame encoding a DLT protein comprising the polypeptide sequence of SEQ ID NO: 4;
(e) a nucleic acid comprising an open reading frame encoding a DLT protein comprising a polypeptide sequence at least 70% identical to SEQ ID NO: 4; and
(f) a nucleic acid comprising a nucleotide sequence that is the complement of any one of(a)-(e).
2. The isolated DLT polynucleotide of claim 1 comprising the nucleotide sequence of SEQ ID NO: 3.
3. A vector comprising the isolated DLT polynucleotide of any one of claims 1 and
2.
4. A host cell which expresses the vector of claim 3.
5. The host cell of claim 4, wherein the cell is selected from the group consisting of animal cell, plant cell, and microorganism cell.
6. A transgenic plant or seed comprising the host cell of claim 5.
7. The transgenic plant or seed of claim 6, wherein the plant is a monocot.
8. The transgenic plant or seed claim 6, wherein the plant is a dicot.
9. The transgenic plant or seed of claim 7, wherein the transgenic plant is rice.
10. A transgenic plant or seed comprising an isolated DLT polynucleotide selected from the group consisting of:
(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleic acid comprising a nucleotide sequence at least 70% identical to (a);
(c) a nucleic acid that specifically hybridizes to the complement of (a) under stringent hybridization conditions;
(d) a nucleic acid comprising an open reading frame encoding a DLT protein comprising the polypeptide sequence of SEQ ID NO: 2;
(e) a nucleic acid comprising an open reading frame encoding a DLT protein comprising a polypeptide sequence at least 70% identical to SEQ ID NO: 2; and
(f) a nucleic acid comprising a nucleotide sequence that is the complement of any one of(a)-(e).
11. The transgenic plant or seed of claim 10, wherein the plant is a monocot.
12. The transgenic plant or seed claim 10, wherein the plant is a dicot.
13. The transgenic plant or seed of claim 11, wherein the transgenic plant is rice.
14. An isolated DLT polypeptide, comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 4;
(b) an amino acid sequence derived from (a) by substitution, deletion and/or addition of one or several amino acids, wherein the amino acid sequence derived from (a) encodes a protein that alters tiller number in a plant; and
(c) an amino acid sequence at least 70% identical to (a) or (b).
15. A method for producing a transgenic plant comprising regenerating a transgenic plant from the host cell according to claim 5.
16. A method for producing a transgenic plant comprising crossing a transgenic plant according to any one of claims 6 and 10 with a non-transgenic plant.
17. A plant produced by the method according to any one of claims 15 or 16 or a transgenic seed derived therefrom.
18. A method of increasing tiller number in a plant comprising expressing an isolated DLT polynucleotide selected from the group consisting of:
(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 ;
(b) a nucleic acid comprising a nucleotide sequence at least 70% identical to (a);
(c) a nucleic acid that specifically hybridizes to the complement of (a) under stringent hybridization conditions;
(d) a nucleic acid comprising an open reading frame encoding a DLT protein comprising the polypeptide sequence of SEQ ID NO: 2;
(e) a nucleic acid comprising an open reading frame encoding a DLT protein comprising a polypeptide sequence at least 70% identical to SEQ ID NO: 2; and
(f) a nucleic acid comprising a nucleotide sequence that is the complement of any one of (a)-(e).
19. A method of producing a dwarf plant comprising expressing an isolated DLT polynucleotide selected from the group consisting of:
(a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3;
(b) a nucleic acid comprising a nucleotide sequence at least 70% identical to (a);
(c) a nucleic acid that specifically hybridizes to the complement of (a) under stringent hybridization conditions;
(d) a nucleic acid comprising an open reading frame encoding a DLT protein comprising a polypeptide sequence of SEQ ID NO: 4; (e) a nucleic acid comprising an open reading frame encoding a DLT protein comprising a polypeptide sequence at least 70% identical to SEQ ID NO: 4; and
(f) a nucleic acid comprising a nucleotide sequence that is the complement of any one of (a)-(e).
PCT/IB2009/055988 2008-12-30 2009-12-29 Genes associated with plant tiller number and uses thereof WO2010076766A1 (en)

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